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NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

Near-infrared Absorption/Luminescence Measurements


A.R. Swamy, J.C. Mason, H. Lee, F. Meadows, M. Baars, L. Strekowski, and G. Patonay Georgia State University, Atlanta, USA

1 INTRODUCTION
The current interest in development of new techniques for analysis is largely attributed to limitations of the more conventional instrumental methods. Classical biomolecule identication usually involves separation of a complex mixture of biological molecules followed by tests for identication of the separated fractions. No single test provides denitive identication of an unknown biomolecule, hence a complex series of tests are required. Such processes are time-consuming and cannot be carried on a reasonable timescale needed in clinical laboratories. These obstacles and the desire to attain adaptability to primitive eld test conditions have prompted many researchers to explore modern alternatives to classical instrumental procedures. Most of these modern analytical tools are characterized by rapid data acquisition and data reproducibility which is due to coupling with computer-aided instrument control, data recording and interpretation. A number of modern instrumental techniques have been applied to the identication of biomolecules. These well-established methods include high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), circular dichroism (CD), gas chromatography (GC), and mass spectrometry (MS). Many of these techniques have also been adapted for eld applications. Most of these techniques conventionally have been applied in the UV/VIS region. However, the NIR region offers several advantages over the conventional UV/VIS region. Sir William Herschel, the English astronomer, discovered the NIR region (600 1100 nm) at the beginning of the 19th century 1 by using a prism and a thermometer; however, this region of the electromagnetic spectrum did not receive much attention until the advent and recent availability of inexpensive light sources, such as diode lasers and detectors such as photodiode and avalanche photodiode (APD) detectors. The 600 1100-nm region corresponds to an energy range of 48 26 kcal mol 1 . Atomic and molecular transitions in this long-wavelength region are processes that require relatively low-energy photons, because the ground- and excited-state species are close in energy. As a result, several classes of molecules, such as polymethine and phthalocyanine (Pc) dyes, and certain elements, such as ruthenium and osmium, require a moderately low energy input to produce spectroscopically measurable electronic transitions. Owing to its sensitivity and selectivity, uorescence is used as a major analytical tool in the identication of target molecules of interest. Typically, this involves using uorophores as reporter molecules or labels. Background uorescence from components other than the

1 Introduction 2 Near-infrared Chromophores and Light Absorption Properties 3 Chemistry of Near-infrared Dyes 3.1 Indocarbocyanine Dyes 3.2 Squarylium Dyes 3.3 Phthalocyanines and Naphthalocyanines

1 3 4 4 7 7 8 9

4 Analytical Applications of Near-infrared Fluorescence 4.1 Applications of Non-covalent Labeling with Near-infrared Dyes 4.2 Bioanalytical Applications of Covalent Labeling with Near-infrared Dyes 4.3 Environmental Applications of Near-infrared Fluorescence 4.4 Other Applications of Near-infrared Fluorescence 5 Conclusions Abbreviations and Acronyms Related Articles References

9 21 24 29 29 30 30

The near-infrared (NIR) region (600 1100 nm) offers several advantages in comparison with the conventional ultraviolet/visible (UV/VIS) region for spectroscopic measurements and detection. The lack of commercial instrumentation impeded the development of NIR techniques for years. The advent of inexpensive photodiodes and diode lasers used widely by the telecommunication industry gave the necessary impetus for development of analytical NIR techniques. This article describes the fundamental properties of NIR uorophores and absorbers and provides a comparison with conventional UV/VIS dyes. The various analytical applications developed in the past decade that have utilized the advantages offered by this region are described with a particular emphasis on bioanalytical uses. Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd

2
Visible fluorophores

ELECTRONIC ABSORPTION AND LUMINESCENCE

Table 1 Comparison of NIR and visible laser excitation


sources Parameter
PAHs Porphyrins

Laser diode 785 100 000C 0.02 0.15 <150

Argon laser 488 3000 15 1800 >5000

Biomolecules

NIR dyes

Wavelength (nm) Lifespan (h) Power output (W) Power consumption (W) Replacement ($)

nm 200 400 600 800 1000

Figure 1 Visible to NIR region and possible interferences from


native uorescence.

uorophore of interest decreases the sensitivity of detection in solution. In biological systems, this background uorescence is typically from the autouorescence of certain biological components. Similar problems are encountered with environmental samples. Typical background uorescence occurs at all wavelengths in the visible region and at various intensities depending on the concentration of interfering molecules present in the sample (Figure 1). Elimination of background uorescence is essential where the undesired components are not separated and uorescence must be detected in the presence of intervening molecules. In uorescence analysis, excitation light can be scattered from interaction with various types of molecules at the surfaces of containers. This scattered light effect is present in all types of uorescence detection. Scattered light contributes to a signicant portion of background noise, especially in biological samples, and it can be due to either Rayleigh, Raman, or Tyndall effects. These interferences can be best minimized by using uorophores with relatively high Stokes shifts (typically >40 50 nm). Fluorescence is more sensitive and selective than absorbance as a spectroscopic tool, not only because it is measured against a zero background but also because the magnitude of uorescence signal, F, at low dye concentrations is expressed by Equation (1): F D 2.303f I0 ebC 1

where I0 is the excitation power, e is the molar absorptivity at the excitation wavelength, f is the quantum yield, b is pathlength, and C is the dye concentration. It can be seen that the limits of detection can be improved by a stronger excitation source. Laser-induced uorescence (LIF) provides a superior approach to improve the sensitivity of uorescence techniques. However, one must keep in mind that the limit of detection increases only as the inverse square root of excitation power and a strong excitation source can cause photo bleaching of

the uorophore. The limitations of conventional lasers as excitation sources include their high price, size, maintenance costs, and their limited wavelength selection. LIF in the NIR region (600 1100 nm) offers several advantages, and the recent advances in semiconductor laser technology have made the use of lasers more practical. In addition, the extensive use of NIR-emitting laser diodes in the telecommunications industry has made them more readily available. These types of lasers are inexpensive (typically <$150), small (1 cm), and have a long operating lifetime (>100 000 h). A comparison of selected visible and NIR laser excitation sources is provided in Table 1. The gallium aluminum arsenide (GaAlAs) laser diode has drawn much interest because its emission wavelength of 785 nm is compatible with several classes of polymethine cyanine and naphthalocyanine (NPc) dyes that exhibit NIR uorescence. 2 In addition to the availability of diode lasers used for excitation, uorescence detection in the NIR region has advantages since noise resulting from scatter is related to wavelength of detection by the factor of 1/l4 . Detection at 820 versus 500 nm results in more than a sixfold reduction in scatter noise. The low background interference in the NIR spectral region allows NIR uorophores to be used as ideal probes in both biological and environmental applications. The advantages offered by the NIR region are summarized in Table 2. Detection in the NIR allows the commonly used photomultiplier tube (PMT) to be replaced with the more efcient photodiode and APD type. The APDs have excellent quantum efciency in the NIR region. Table 3 illustrates the advantages offered by APDs in comparison with PMTs. These benets include their low cost, compact structure, and their durability. In addition, they have low internal noise and very low power consumption. All these features allow NIR uorescence Table 2 Comparison of NIR and visible regions
Noise source Detector Scatter (Rayleigh/Raman) Background/autouorescence NIR region Low Reduced Mostly absent Visible region High 6 greater at 520 nm than 820 nm Autouorescence of biomolecules

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

Table 3 Comparison of APDs and PMTs


Parameter Replacement cost ($) Lifetime (h) Quantum efciency (at 820 nm) (%) Internal amplication Size Power consumption APD <50 >100 000 80 High Very small (nm) Very low PMT 500 10 000, sensitive to overexposure 0.3 Low Small (cm) Low

advantages offered by this novel methodology ranging from DNA sequencing, pH and hydrophobicity determination, metal ion detection, antibody (Ab) labeling, HPLC, and high-performance CE.

2 NEAR-INFRARED CHROMOPHORES AND LIGHT ABSORPTION PROPERTIES


To take full advantage of the high-technology NIR instruments, new NIR chromophores with specic properties are required. The comprehension of the color structure relationship of near-infrared dyes (NIRDs) can provide useful information for their development since the lightabsorption property of an organic molecule is correlated with its structural features. A highly conjugated system in either a linear or a cyclic arrangement in the molecule is responsible for the absorption maxima in the NIR region. In principle, the absorption property of a chromophore is the characteristic of the energy that is absorbed to cause the electronic transition. NIR-absorbing chromophores require relatively low energy for the transition, and this corresponds to the longer wavelength of the electromagnetic spectrum in comparison with UV/VIS absorption. Since the transition energy is the energy required for a single electron to be excited from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), the closeness between the HOMO and LUMO orbital is primarily responsible for the amount of energy required to induce this electronic transition. Therefore, the shift of the absorption maxima (lmax ) into the NIR region can be induced if the gap between the HOMO and the LUMO orbitals is brought close enough to give the transition energy in the range 48 26 kcal mol 1 . In practice, effective structural modication of the chromophore can cause the desired bathochromic shift. Grifths summarized in detail the general strategies to develop new NIRabsorbing dyes using this approach. 3 These strategies include (i) extending the conjugation of a chromophore, (ii) increasing the interaction between electron donor and acceptor groups within a chromophore, (iii) altering the electronegativity of atoms within a chromophore, (iv) introducing specic branching or bridging within a chromophore, (v) metal complexation with a chromophore, (vi) intermolecular charge-transfer complex formation, and (vii) formation of a free radical that is part of the chromophore. The Pariser Parr Pople molecular orbital (PPPMO) method 6 and XNDO/S method 7 are useful in the estimation of the absorption position of the chromophore that needs to be constructed. The perturbational molecular orbital (PMO) theory in the form of Dewars rule

instrumentation to be highly versatile and amenable to miniaturization. These features aid in the development of a portable compact and rugged instrument for eld applications. The recent progress in solid-state diode laser technology, the availability of commercial NIR absorbing dyes, and the development of diverse synthetic routes to functionalized NIR absorbing dyes have allowed NIR spectroscopy to be used in many diverse applications. These specic uses rely on the response of the chromophore after its excitation by NIR radiation. A simple absorber can be used in spectrophotometric analysis, 3 as molecular probes in immunoassays (IAs), or in applications of security printing, such as laser-readable bar codes. 4 The absorber can re-emit the input energy via uorescence or phosphorescence. Also, the NIR-absorbing chromophore can convert the excited-state energy into other forms that can be utilized for specic applications. After absorption of laser energy, the chromophore can produce a local heating effect in the medium in which it resides. 3,4 This technique is used in the optical data recording technologies, such as WORM (write once read many) and DRAW (direct read after writing), 4 full color laser imaging, and in medicinal uses such as tissue welding. 3 Conversion of the NIR radiation into electrical energy by the chromophore is the fundamental basis for photovoltaic cells and electrophotography, including laser printing. 3,4 Furthermore, transfer of the chromophores excited-state energy to endogenous ground-state molecular oxygen is the foundation for the selective destruction of neoplastic tissues in photodynamic therapies. 4,5 In particular, the NIR region provides a convenient spectral range for photodynamic therapies and in vivo imaging owing to the multicentimeter penetration of biological tissues. 5 Taken together, these benets make NIR analysis an operator-safe, nondestructive, sensitive, and versatile technique that is taking the place of conventional methods, such as radiolabeling. Moreover, dilute samples can be analyzed safely and relatively fast compared with other techniques. Several applications have been developed in the past decade utilizing the various

4
0.043 0 0.032 0.045
+

ELECTRONIC ABSORPTION AND LUMINESCENCE


0.024 0.003 0.024 0.003 0.043 0

S
Z *

0.11 0.123 0.11

S N* Et

N 0.25 0.06 * Et

0.045 0.032

0.25 0.06 *

(1)

R * * * *

S N* Et

N Et

(2)
R = H, max = 758 nm R = CN, max = 860 nm ( = 102 nm) R = OMe, max = 730 nm ( = 28 nm)

chromophores but also other characteristics for different analytical applications are desired in the design of these systems. NIR chromophores with proper functional groups, organic and/or water solubility, high molar absorptivity or uorescence maximum in the NIR region are required for most applications, including medical and optical purposes, dye lasers, and photographic sensitizers. These characteristics can be generally introduced in two ways, namely (i) by the structural modication of commercially available dyes and (ii) by direct synthesis utilizing appropriate precursors. Several examples of direct synthetic routes to NIRDs with specic applications are discussed below. 3.1 Indocarbocyanine Dyes

can also aid in the prediction of the light-absorption properties of chromophores with structural changes. 8 This rule is particularly useful in the development of new NIRDs since one can correlate the spectroscopic effects with a structural change at a specic position. In the basic analysis of an odd-alternate system of the conjugated chromophore, each alternate atom starting from a terminal donor is starred, as exemplied in (1). Dewars rule implies that an increase in the electronegativity at a starred carbon results in a hypsochromic shift. On the other hand, a bathochromic shift will be induced if the electronegativity of an atom at unstarred position is increased. In particular, a bathochromic shift of a cyanine chromophore is induced if the electron donor groups at starred positions or electron-withdrawing groups at unstarred positions are introduced. 6 This is further illustrated by the studies of substituted trimethine cyanines (1) on the basis of the PPPMO theory by Yasui et al., which showed the decrease in the electron densities at the starred positions and increase at the unstarred positions of the polymethine chain accompanying the rst excitation of (1). 9 An additional example of the application of Dewars rule is given in (2) for three heptamethine cyanine dyes of a general structure (2). As can be seen, the electron-withdrawing substituent (CN) at the central unstarred position of the chromophore causes a bathochromic shift and a hypsochromic shift is observed for the electron-donating group (OMe) relative to absorption of the parent unsubstituted dye R D H). 10

3 CHEMISTRY OF NEAR-INFRARED DYES


So far, the theoretical correlation between structure and absorption properties has been discussed. These general rules can also be applied in the practical synthetic design of NIR-absorbing dyes. However, it should be noted that not only the light absorption properties of NIR

Functionalized NIR absorbing chromophores are widely used for labeling purposes. However, the commercial availability of such systems is somewhat limited. The rst NIR-absorbing dyes containing NDCDS and C(O)CH2 I functionalities for selective coupling with amino and thiol groups, respectively, of biomolecules were synthesized by Waggoners group. 11 These dyes, synthesized by using classical chemistry, contained the reactive group at one of the terminal heterocyclic units, and were extremely difcult to purify. Subsequently, Strekowski et al. 12,13 discovered a facile functionalization of indolium heptamethine cyanine dyes at the central meso position of the chromophore, and the synthesis of (8a c) is given in Scheme 1 as an example. These dyes are substituted with an isothiocyanato or N-succinimidyl ester function for selective labeling at the amino group of proteins or bioconjugates such as amino-functionalized nucleotides. 13,14 The structural design of (8) includes the presence of sulfonic acid groups for excellent solubility in water and the overall symmetry of the molecule to contribute to the facile purication of the dye by simple crystallization. The symmetry is responsible for a high molar absorptivity of the chromophore. An important structural feature is the rigid trimethylene bridge in the center of the molecule that enhances the uorescence quantum yield by decreasing conformational freedom of the NIR uorophore in comparison with that of the uorophore without the bridge. The key synthetic step to (8) is a facile nucleophilic displacement of the chloro substituent in the intermediate product (7) which, apparently, involves an SNR 1 pathway. 12 In the preparation of (7) the starting material (6) is derived from dialdehyde (11) (for structure, see Scheme 2) by the reaction with aniline. The second component (5) is prepared efciently by the reaction of (3) and (4), as shown in Scheme 1. This approach, as discussed above, was utilized by Flanagan et al. 15 in DNA sequencing involving capillary

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

H3C N

CH3 CH3 +

O O

H3C
i

CH3 CH3

N+

(3)

(4)
Cl Ph SO3

Cl
+

N H

N H

Ph

(5)
ii

(6)

CH3 CH3 N+
O 3S

H3C H3C N SO3Na


iii

CH3 CH3 N+

Cl

H3C H3C N

(8a-c)
a X = S, R = NCS b X = O, R = NCS O O c X = O, R = CH2CH2CO N O

3S

(7)

SO3Na

Scheme 1 Synthetic route to heptamethine dyes. (i) Reux in 1,2-dichlorobenzene. (ii) Reux in EtOH in the presence of sodium
acetate. (iii) Substituted sodium phenolate or thiophenol in DMF, 23 C. [Strekowski et al. 13 approach.]

X O N+
O S 3

Y N SO3

(9ag)
X, Y a H, I b H, Br c H, Cl d H, F X, Y e Br, Br f Cl, Cl g H, H

gel electrophoresis (CGE). Their dyes (9a g) contain different atoms incorporated in the periphery of the chromophore, and have been prepared by substitution of the meso-chlorine atom in the intermediate dye with 2,5-disubstituted phenolates. The introduction of uorine or heavy atoms such as iodine, bromine, and chlorine into the molecular framework has been found to affect the uorescence lifetime without affecting the spectral properties of the chromophore. It was reasoned that introduction of heavy atoms induces spin orbit coupling. As a result, the intersystem crossing (ISC) rates are enhanced producing a reduction in

the excited-state lifetimes associated with the singlet state. Recently, several commercial NIR-absorbing cyanine dyes have also been made available with amine-reactive groups for covalent coupling with synthetically altered nucleotides or oligonucleotides. The dyes are available under the Cy and IRD trade names, and have been synthesized by Mujumdar et al. 16 and LI-COR, Inc., 17 respectively. The LI-COR IRD dyes, such as the nonsymmetric heptamethine dye IRD800 phosphoramidite [(18), Scheme 2], can be prepared by using the methodology described by Narayanan et al. 18 The synthetic route involves heating the bisaldehyde (11) and the hydroxy-functionalized quatenary indolium salt (10) with azeotropic removal of water (Scheme 2) to give the intermediate half cyanine carboxaldehyde (12). This intermediate generated in situ is further allowed to react with indolium alkyl sulfonate (5), which furnishes the cyanine dye (13). The absorption maximum of the NIR chromophore in (13) can then be ne tuned by addition of an electron-donating or electron-withdrawing group at the meso-position, as already discussed. Thus, the reaction of (13) with sodium phenolate (14) furnishes a phenoxy derivative (15). Further treatment of the meso-substituted cyanine dye (15) with 1H-tetrazole (16) and 2-cyanoethyl tetraisopropylphosphorodiamidite

6
Cl + N+ Br HO CHO
i

ELECTRONIC ABSORPTION AND LUMINESCENCE

Cl

(11)

CHO

OH

HO

(10)

(12)

N+

SO3

(5)

O N
+

ii

Cl N
+N

N SO3

ONa

(14)
HO

SO3

OH

(15)

(13)

H N N N

N N

O
+

(16)
N P O CN N

N SO3

(17)

P O

CN

(18) IRD800 phosphoramidite

Scheme 2 Synthetic route to IRD800 phosphoramidite. (i) Reux in a mixture of benzene and n-butanol with azeotropic removal
of water. (ii) Stirring in DMF at 23 C.

(17) produces the nal dye (18) (IRD800 ) that contains a reactive phosphoramidite functionality for automated DNA synthesis applications. The spectral properties of IRD800 are retained after coupling with the oligonucleotide. The unlabeled chromophore (18) exhibits an absorption maximum at 787 nm in methanol with a 25-nm Stokes shift in uorescence and a 15.0% quantum yield, while the oligonucleotide-labeled chromophore (18) has

an absorption maximum at 796 nm in water with a 23-nm Stokes shift and a 14.7% uorescence quantum yield. Using similar synthetic strategies, nonsymmetric pentamethine cyanine dyes have been developed by Mujumdar et al. 16 The dyes, such as Cy5-dUTP (19), can be covalently linked to biomolecules including nucleotides and the conjugates used in enzymatic polymerization

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

processes. The dye-labeled dUTP analogs can be incorporated into DNA probes by nick translation, random priming, and polymerase chain reaction (PCR).

O3S

SO3

N+

(absorbance, emission, and lifetime) are independent of pH under physiological conditions (pH 6 9). 19 As shown in Scheme 3, Terpetschnig et al. 19 synthesized a water-soluble NIR-absorbing dye containing an activated ester moiety. The reaction of (20) and (21) 20 produced carboxy-substituted dye (22). Following purication by preparative thin-layer chromatography (TLC), the nonsymmetric squaraine (22) was coupled to N-hydroxysuccinimide to produce the active ester (23). 3.3 Phthalocyanines and Naphthalocyanines

O NH

O NH N O O OH O O O O P O P O P O O O O

(19) Cy5-dUTP

3.2 Squarylium Dyes In addition to the indocarbocyanine dyes, synthetic routes to indolenine squaraine dyes, such as the N-succinimidyl ester derivative (23) (Scheme 3) for conjugation to proteins, are being developed. The squarylium dyes exhibit high photostability and quite long uorescent lifetimes (nanosecond range) in aqueous solutions. Further benets of using NIR-absorbing squaraine dyes for biological applications include the fact that their spectral properties

The NIR-absorbing dyes such as Pc and NPc type are used as industrial colorants and in special applications of electronics and optics. 21 They efciently complex various metal ions, and approximately 70 central metal ion species of Pcs are known. The cation is held in the planar 18electron cavity and the metal cation can strongly inuence the physical and spectral properties of the dye, including absorbance, emission, and uorescence lifetime. 21 These NIRDs are sensitive to environmental changes such as pH and metal concentration. Classical synthetic routes to metal ion-containing Pc dyes [metal ion phthalocyanine (MPc) dyes] are illustrated in Scheme 4 where the metal ion acts as a central template for cyclotetramerization. As can be seen, MPc (28) is obtained directly from diiminoisoindolenine (24), phthalic anhydride (25), phthalonitrile (26), or phthalimide (27) in the presence of a metal salt. 21 These dyes, though highly insoluble, are extremely stable and can be obtained by precipitation and further puried by sublimation. Metal ion-containing
HOOC OH
+N

HOOC
+N

HO + Cl

O O
i

Cl

N O

SO3

(20)

(21)
ii

3S

(22)

O N

O O

O Cl OH
+N

N O

3S

(23)

Scheme 3 Synthetic route to nonsymmetric squarine dye with N-succinimidyl moiety. (i) Reux in n-butanol and toluene with
azeotropic removal of water. (ii) Stirring with N-hydroxysuccinimide and DCC in dimethoxyethane at 23 C. 19

8
NH NH NH
i

ELECTRONIC ABSORPTION AND LUMINESCENCE

(24)
O O O

ii

N N
i

N M N

N N N

(25)
CN CN

One approach to overcome the insolubility is to incorporate sulfonate groups into the periphery of the existing Pcs or NPcs (Scheme 5). Patonay et al. at Georgia State University 22 have synthesized a tetrasulfonylated aluminum NPc (31) that can be incorporated into an optical probe for NIR metal determinations. Once incorporated into the ber, the four sulfonate groups at the periphery of the NIR uorophore interact with metal ions, such as KC , to produce spectral changes in their absorption maximum (lmax ) and/or uorescence intensity. The NIR probe studied by Patonay et al. provides metal detection over the range 1 10 8 0.5 10 1 M.

(26)
O NH O
ii

(28)

4 ANALYTICAL APPLICATIONS OF NEAR-INFRARED FLUORESCENCE


LIF offers tremendous advantages over conventional analytical methods. The NIR region which has no background interference when coupled with LIF can attain sensitivities obtained by radiolabeling. This method also provides a step towards the maximum achievable sensitivity, that is, single-molecule detection. Many reviews of NIR uorescence that describe dyes, instrumentation, and applications are available. An authoritative review, though dated, is provided by Warner et al. 23 A recent book summarizing the results of a North Atlantic Treaty Organization (NATO) Advanced Research Workshop covers the synthesis, optical properties and applications of selected NIRDs in hightechnology elds. 24 In the next section, representative examples are reviewed to emphasize and acknowledge further the advantages and usefulness of NIR uorescence techniques.
SO3

(27)

Scheme 4 Classical synthetic routes to metal ion NPcs. (i) Heat


in a high-boiling solvent (e.g. quinoline) with metal salt. (ii) Heat in a high-boiling solvent with urea and metal salt.

NPcs, such as (31) in Scheme 5, are obtained in a similar fashion. The inherent sensitivity to metal and hydrogen ion concentration makes Pcs and NPcs ideal candidates for use in pH or metal determination. However, the poor aqueous solubility is a shortcoming that has to be overcome by structural modication or functionalization.

N CN CN
i

N Al N

N N Cl N
ii

N O3S N N

N Al N

N N Cl N SO3

N N

(29)

SO3

(30)
oleum (15% of free SO3 ). 22

(31)

Scheme 5 Synthesis of tetrasulfonylated aluminum NPcs. (i) Reux in 1,2-dichlorobenzene containing 1 M AlCl3 . (ii) Heat in

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

4.1 Applications of Non-covalent Labeling with Near-infrared Dyes The NIRDs used as reporter molecules can be attached to the biomolecule of interest by two methods, via (i) covalent bond and (ii) noncovalent hydrophobic and/or ionic interactions. Theoretically, the hydrophobic backbone of many NIRDs allows for noncovalent labeling of large biomolecules. However, only a limited number of practical applications have been tested.

et al. 27 in terms of stability and specicity. Their results indicated that noncovalent labeling occurs rapidly at a physiological pH range. However, this interaction was found to be nonspecic and less stable than covalent binding. 4.2 Bioanalytical Applications of Covalent Labeling with Near-infrared Dyes Many bioanalytical applications which rely on covalent labeling have been developed. Techniques that utilize this methodology include IAs, CE, DNA sequencing, and the development of gene probes. 28 This section details some of these applications with their associated merits and disadvantages. 4.2.1 Application of Near-infrared Fluorescence in Capillary Electrophoresis Analytical separations of intact proteins using HPLC and CE have been shown to have excellent resolving power. 29 CE is particularly advantageous for protein analysis because of its higher chromatographic efciency, faster separations time, and ability to use small amounts of material. The low molecular diffusion rates of proteins help achieve theoretical plate numbers as high as 106 . Many excellent reference books on CE have appeared in recent years, 30 32 and the reader is encouraged to refer to them for additional information not covered below. The Handbook of CE 31 is very thorough, while the CE primer series offered by Beckman Instruments 30 provides a brief overview of the principles of different separation techniques for a variety of applications. Five main modes of electrophoresis have been developed over the years. These include capillary zone electrophoresis (CZE), isoelectric focusing (IEF), CGE, isotachophoresis (ITP), and micellar electrokinetic chromatography (MEKC). These different modes account for the wide applicability of CE to different analytes. However, one of the challenges in CE applications is the detection of analytes at low concentrations. This is a result of the physical dimensions of the capillary used for separation. Typical injection volumes in the nanoliter range combined with small detector windows of approximately 100 200 mm require highly sensitive detection systems. UV/VIS absorbance is the most commonly used detection method for CE. The advantage is simplicity, low cost, and ease of use. Detection limits are in the range of 10 13 10 16 mol for direct absorption (a factor of 10 less for indirect methods). Other methods employed include MS, electrochemical, refractive index and radiometric detection. 28 Fluorescence detection can greatly enhance the sensitivity of CE methods. For example, direct uorescence provides detection limits in the range of

+N

NaO3S

SO3

(32)

Cl N I
+N

(33)
NCS Cl N
+N

SO3

(34)

X N R1
+N

R2

(35)
X = OPh-4(NCS), SPh-4(NCS); R1 = Et, (CH3)4SO3Na; R2 = Et, (CH3)4SO3

One of the earliest applications studied was the binding of indocyanine green (ICG) (32) to serum albumins by Kamisaka et al. in 1974. 25 Similar studies by Sauda et al. 26 using semiconductor laser uorimetry illustrated that human serum albumin labeled with ICG provided picomolar detection limits. A comparison of noncovalent [(33) and (34)] and covalent dyes (35) labeling used in HPLC determination was investigated by Williams

10
10 15 10 17 mol (a factor of 10 less for indirect methods). However, the number of compounds that uoresce naturally is limited and derivatization of analytes with a uorescent tag is frequently required. Fluorescence emission is measured against a zero background signal, resulting in improved detection limits in comparison with absorption methods. Furthermore, LIF coupled to CE provides a sensitivity of approximately 10 18 10 23 mol. This improved sensitivity of uorescence detection is attributed to concentrating high power monochromatic light into a very small area, which provides an ideal excitation source for CE uorescence detection. LIF is one of the most sensitive methods of detection available for use with CE. Although the number of publications on the subject has been rising steadily each year, the practical use of LIF with CE has been limited owing to scatter from incident light reected by the capillary walls with on-column LIF detection. Scatter can be greatly reduced by utilizing postcolumn detection using a sheath-ow arrangement, rst described by Chen and Dovichi. 33 However, this set-up adds a signicant amount of complexity to the method, requires additional equipment such as a low-ow HPLC pump, and is not commercially available. Another deterrent is the high cost associated with visible lasers which are suitable for excitation of the more popular uorescent labels including uorescein isothiocyanate (FITC) and 3-(4carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA). Several authors have reported on the advantages of using semiconductor lasers with CE. Most investigations to date have involved the use of far-red uorescent dyes. Williams et al. 34 described a diode laser-based indirect absorbance detector for the analysis of a series of tetraalkylammonium ions. In this study, rhodamine 700 (labs D 642 nm, lem D 668 nm) was used as a background absorber, and detection limits were found to be of the order of 20 mM. Another study utilizing a time-resolved photon counting uorimetry CE method was described by Song et al. 35 Four uorescent dyes were evaluated using an LIF detector with a 74-ps pulsed semiconductor laser emitting at 655 nm. The dyes investigated included methylene blue (labs D 665 nm, lem D 685 nm), oxazine 725 (labs D 655 nm, lem D 679 nm), 1,10 ,3,3,30 ,30 -hexamethyl dicarbocyanine (HIDC) iodide (labs D 636 nm, lem D 667 nm), and rhodamine 700. The lowest limit of detection was reported to be 2 fmol, which was obtained for oxazine 725 at a concentration of 0.2 10 5 M. Fuchagami et al. synthesized a novel far-red uorescent labeling dye for use with a semiconductor LIF detector. 36 Several amino acids were derivatized with 9-cyano-N,N,N 0 -triethylN 0 -(50 -succinimidyloxycarbonylpentyl)pyronine chloride (labs D 663 nm, lem D 685 nm) and were separated and detected by CE. The experimental system used a 2-mW

ELECTRONIC ABSORPTION AND LUMINESCENCE

Table 4 Comparison of visible (R6G) and NIR (IR132) single-molecule detection. [Adapted from Soper et al. 38 ]
Parameter Excitation wavelength (nm) Observed wavelength (nm) Instrument response (fwhm) (ps) Photon detection efciency Probe volume (pL) Transit time, tt (ms) Background rate in time window (counts s 1 ) Average photons/molecule Detection efciency (%) Probability of error Visible 532 570 100 0.0007 1.1 24 225 39 78 0.3 NIR 800 840 250 0.007 0.8 10 145 (12) 18 (4) 97 (7) 0.04

semiconductor laser emitting at 660 nm and a PMT detector. Detection was performed off-column using a sheath ow cell to reduce Rayleigh scatter, achieving a limit of detection [signal-to-noise ratio (SNR) D 2] of 800 zmol. The advantages of using APD CE detectors at wavelengths above 650 nm have been reported. Kawazumi et al. 37 compared PMT and APD detection using oxazine 725 excited by a 29-mW semiconductor laser emitting at 655 nm. The APD was operated in a nearGeiger mode with time-gated detection, resulting in a 47-amol limit of detection. The APD limit of detection was 98 times greater than that with a PMT. As already mentioned, detection in the NIR region offers considerable advantages over working in the conventional UV/VIS region. A signicant amount of work with NIR uorescent dyes including their applications to CE has been conducted at Louisiana State University by Soper et al. 38 40 They observed bursts of photons from single NIR uorescent molecules in a owing stream using a Ti : sapphire laser (12 mW) and an APD detector. The dye infrared (IR)132 (labs D 805 nm, lem D 847 nm) at a concentration of 25 fM in methanol was used in these experiments with time-gated off-column detection. 38 A comparison of a single-molecule detection of visible (R6G) dye and the NIRD IR-132 (Table 4) shows a signicant reduction in background uorescence, resulting in a lower discriminator threshold and error probability, and thereby provided a higher single-molecule detection efciency in the latter case. A set-up similar to the photon burst experiment, using continuous excitation and CE with on-column detection, was utilized for an investigation of binary solvent effects in CZE with several NIRDs and dye-labeled amino acids. 39 One of the dyes substituted with an isothiocyanate group (37), was synthesized from dye (36) by Soper et al. and covalently bound to amino acids. The results showed that the detectability, efciency, and resolution of the

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

11

Cl N
+

N SO3

3S

(36)
NCS

O N
+

N SO3

3S

(37)

Table 5 SNR, theoretical plates per meter and mass detection


limits for cationic dyes in mixed methanol aqueous borate buffers (pH 9.1). [Adapted from Flanaghan et al. 39 ] Dye SNR 95 : 5 DTTCI IR-140 IR-132 HDITCP 1478 1713 1636 1858 60 : 40 146 247 Plates m 95 : 5 65 500 64 500 64 300 57 800
1

Mass detection limit (ymol) 95 : 5 5460 446 429 351 60 : 40 20 280 24 960

60 : 40 63 600 55 700

HDITCP, 1,10 ,3,3,30 ,30 -hexamethyl-4,40 ,5,50 -dibenzo-2,20 -indotricarbocyanine perchlorate.

NIR methodology can be signicantly improved with the use of mixed organic aqueous running buffers (Table 5). However, cationic NIRDs were found to be problematic at low concentrations owing to capillary wall adsorption. The on-column limit of detection was found to be 0.4 zmol using the dye HDITCP (labs D 780 nm, lem D 825 nm) at a concentration of 2 10 10 M in 95% methanol, and the limit of detection of a dye-labeled amino acid was 21 zmol, using a 1 10 9 M solution in 9 : 1 methanol water buffer. A comparable CE system with a 10-mW Ti : sapphire laser emitting at 795 nm was used to evaluate NIR uorescence detection for CGE and DNA sequencing applications. A comparison between detection for NIRD-labeled primer (38) and uorescein labeled primer (39) is shown in Table 6. Under similar conditions, the signicantly lower Raman contributions and background uorescence resulted in a background of 10 000 counts for NIR compared with >200 000 counts for visible excitation in contrast to the larger detection efciency at 800 nm in comparison with 550 nm. Although the NIRD exhibits a lower

quantum efciency, a much lower limit of detection of 34 zmol was achieved for an IRD41 labeled M13 primer (labs D 786 nm, lem D 812 nm) in comparison with 1.5 amol for the visible primer primarily owing to the signicantly smaller background in the NIR region. 40 Until recently, all near-infrared laser-induced uorescence (NIRLIF) work completed to date involved the use of laboratory-made CE systems and detectors. However, two commercial LIF detectors are currently available for use with CE, manufactured by Beckman and Zeta Technology. The Beckman LIF detector is specically designed for use with their P/ACE CE instrument, while the Zeta LIF detector is a general-purpose detector also applicable to HPLC use. Both detectors are optimized for use in the visible region and utilize PMT detectors, with laser excitation via a ber-optic cable. Recently, Patonay et al. at Georgia State University, actively involved in research in other areas of NIR uorescence, developed a simple interface between a commercial APD-based NIRLIF detector and a CE instrument. The system retained the fully automated injection, separation, and data collection capabilities of the commercial CE instrument and was optimized for detection around 820 nm with laser excitation at 787 nm, where the detector sensitivity was improved about 400-fold in comparison with the conventional PMT-based LIF detector. To illustrate the challenges in design of developing a NIR uorescence detector interface to exploit fully the advantages offered by this region, a more detailed description of the work done by Baars et al. 28 is presented in the following section. A Beckman LIF detector with the P/ACE 5000 CE instrument was modied to accommodate the diode laser excitation and NIR uorescence detector. The back mounting plate and a portion of the self-aligning beam probe assembly of the Beckman LIF detector was used in the interface. The at mirror, lter holder, PMT and detector electronics were removed resulting in a collimated uorescence output from the LIF cartridge, passing through the beam block and beam probe, to allow interfacing with the NIR detector. A proprietary microscope and laser assembly manufactured by LI-COR, Inc. (Lincoln, NE, USA) was used as the NIRLIF detector. This detector system is comparable to components used in the commercially available automated DNA sequencing instrument 41 (LICOR Model 4000), with the exception of modied focal length optics and an extra band-pass lter. The laser assembly for excitation contained a GaAlAs laser diode emitting around 787 nm (20 mW peak power, modulated with a 50% duty cycle) and a focusing lens (focal length f D 46 mm). The detector consisted of a three-stage Peltier-cooled APD. The detector assembly contained a plano-convex lens (f D 31 mm) to collect the uorescence image, three identical band-pass lters (825 15 nm)

12

ELECTRONIC ABSORPTION AND LUMINESCENCE

O N
+N

S NH

NH

SO3

O P O

O TGACCGGCAGCAAAATG

(38)
O O O P TGACCGGCAGCAAAATG O S N H NH

COO

OH

(39)

to reduce background noise from laser scattered light (Rayleigh), and a second plano-convex lens (f D 31 mm) to refocus the signal on to the APD photoactive area (0.5 mm diameter). The interface between the LIF cartridge and the NIR uorescence detector comprised an aspheric condenser lens mounted immediately past the beam probe assembly. An 800-m circular aperture was installed at the focal point of the condenser lens to reduce stray light. The NIR detector was xed to an x y z micrometer stage installed on the mounting plate and focused on the image produced by the interface lens. The diode laser was disconnected from the LI-COR microscope assembly and was mounted on another x y z micrometer stage mounted on the side of the CE base. The laser signal was focused directly on the excitation ber, resulting in a 4 mW average excitation power at the capillary interface. The optical path of the complete system is shown in Figure 2. 28 Fluorescence emission was collected by the mirror and reected as a collimated beam at 180 from the angle of excitation and the collimated beam was then focused by the aspheric condenser lens to the appropriate image size, at the focal length of the detector. The detector optics lter the uorescence signal through three band-pass lters prior to focusing the signal on the window of the APD detector. Instrument control

Table 6 Electrophoretic parameters and detection limits for NIRD-labeled primer and uorescein-labeled primer. [Adapted from Williams and Soper 40 ]
Parameter Migration time (s) Apparent mobility (cm2 V 1 s 1 ) Injection volume (L) Amount injected (mol) Net signal (counts s 1 ) Background (counts s 1 ) SNR Detection limit (mol) (SNR D 3) Quantum yield of tag NIR primer 2522 5.6 10 2.9 10 3.8 10 33 500 10 000 335 3.4 10 0.07
5 9 19

FITC primer 660 2.1 10 1.1 10 5.8 10 15 920 19 490 114 1.5 10 0.90
4 8 17

20

18

and data collection of the P/ACE 5000 CE system were fully automated through the Beckman System Gold chromatographic software (version 8.01), run on a 486-33 personal computer. The detector signal was interfaced with the chromatographic software. This was accomplished by connecting the analog detector output to a Beckman 406 analog-to-digital convertor (ADC), whose output can be collected by the chromatographic software. In their set-up, the P/ACE 5000 CE unit was used to control all CE functions such as capillary rinses and injection and separation parameters and in addition, it was

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

13

LIF cartridge Capillary Diode laser Excitation fiber 0.8 mm slit Lens 1 Lens 2

Mirror

APD detector Aspheric condenser lens Interface Band-pass filters LI-COR detector

Inlet

Outlet

Figure 2 Optical path of NIR/LIF interface with CE instrument. used for data collection and chromatographic integration, reporting, and plotting functions. The maximum detector output was matched to the 2-V input limit of the ADC using a simple voltage divider circuit. A block diagram of the main instrument components is shown in Figure 3. The image size produced by the aspheric condenser lens was evaluated for proper matching of the image size to the photodiode active surface area. The experimentally determined mean diameter is 0.575 mm, indicated a slight overlling of the 0.5-mm detector window. The rapid change in signal upon minor detector position was a good indication of an acceptable image size match. A three-dimensional representation of the image produced by the aspheric condenser lens is shown in Figure 4. The sensitivity of the system was evaluated by injecting serial dilutions of the labeling dye NN382 (40), (labs D 776 nm, lem D 796 nm, H2 O) which is suitable for use with the systems excitation and detection wavelengths (Figure 5). A linear detector response (r D 0.9999) was observed over more than a 250-fold concentration range between 1.4 10 9 and 5.5 10 12 M (Figure 6). A SNR, signal/root mean square noise) of 15 was observed for a 70-nL injection of the 5.5 10 12 M solution, which indicated a limit of detection (SNR D 3) of approximately 80 zmol. The electropherogram of a 70-nL injection of the 5.5 pM solution (385 zmol injected) is provided in Figure 7. The sensitivities obtained were under 100% aqueous conditions, and additional improvements could be expected with the use of organic modiers, cyclodextrins, or micellar additives above their critical micelle concentration in the run buffer, from an improved uorescence quantum yield when the dye is exposed to a more hydrophobic environment. Comparison of the photophysical properties of the dye in methanol suggested that a fourfold improvement in signal could be obtained when using the dye NN382 in a more hydrophobic run buffer, containing a cosolvent such as methanol. Additional improvements in detection sensitivity could be attained using an NIRD with an emission wavelength better matched to the band-pass lters in the detector. The current system utilizes three 825-nm band-pass lters that signicantly attenuate the uorescence signal emitted at 800 nm. The relative uorescence signal reaching the detector is only a fraction of the uorescence produced. Light collection efciency and lens and lter transmission efciency inuence the amount of light that will

406 A/D converter

486-33 PC System gold software Capillary cartridge

Detector electronics

APD detector Diode laser

P/ACE 5000 CE

Figure 3 Experimental instrument component diagram.

14
Image

ELECTRONIC ABSORPTION AND LUMINESCENCE

100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0

X-a

xis

0.2

0.0 0.4 0.4 0.6 0.8 1.0 0.6 0.8 0.2

(mm

is ( Y-ax

mm

Figure 4 Three-dimensional representation of the image produced by the aspheric condenser lens.
NCS NaO3S O N
+N

0.10 0.09 776 nm 796 nm Excitation Emission

Respo nse
1000 900 800 700 600 500 400 300 200 100 0

SO3Na

0.08

0.07 0.06 0.05 0.04 0.03 0.02 0.01

NaO3S

SO3

(40)

0.00 700 720 740 760 780 800 820 840 860 880 900

actually reach the detector. Thus, the maximum efciency of the optical path in the experimental set-up can be estimated as follows: light collection efciency of mirror: 10%; transmission efciency of aspheric condenser lens: 98%; transmission efciency of rst detector lens: 98%; maximum transmission efciency of each band-pass lter: 70%; transmission efciency of second detector lens: 98%.

Wavelength (nm)
Figure 5 Absorbance and uorescence spectra of NN382 (in phosphate buffer, pH 7.2). reduces the number of photons that actually produce a detector response. The improvement that can be attained when better matching band-pass lters with the dye emission prole, or dye selection with the system band-pass lters, can be estimated. A comparison made between the area of the NN382 (40) emission curve from 810 to 840 nm and the maximum area that could be attained from 781 to 811 nm (15 nm from the emission maximum) shows that maximum area was 70% larger than the area of the curve corresponding to the band-pass lter wavelengths. Using a dye with an emission wavelength

The overall result is that only about 3.2% of the uorescence signal reaches the detector. The quantum efciency of the detector (even though relatively high for an APD at 800 nm, 80%) further

Fluorescence response

Absorbance

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

15

100 Peak area response 90

Peak area response

80 70 60 50 40 30 20 10 0

20 15 10 5 0 100 200

Concentration (pM)

100

200

300

400

500

600

700

800

900 1000 1100 1200 1300 1400

Concentration (pM)
Figure 6 NN382 response curve (384 98 zmol on-column).

Fluorescence response (V)

0.009 0.007 0.005 NN-382 0.003 0.001 0.001 0 1 2 3 4 5 6 7 8 9 10 11

Time (min)
Figure 7 Electropherogram of 70-nL injection of 5.5 10
NN382.
12

around 825 nm could therefore result in about a 1.7-fold increase in signal, assuming a comparable uorescence emission prole. The noise would remain unchanged, suggesting that an additional 1.7-fold improvement in SNR could be attained. All the above-mentioned factors demonstrate important aspects of LIF instrumentation design. To optimize sensitivity, laser excitation should occur below the absorption maximum when a dye has a relatively short Stokes shift. Band-pass lters should be optimized for the uorescence emission maximum of the dye. This design will effectively attenuate the Rayleigh scatter from the laser while optimizing uorescence signal collection. The automated NIRLIF system developed has sensitivity in line with state of the art results obtained by several investigators utilizing highly optimized laboratory-made detection systems. The latter systems utilize high-quality microscope optics for uorescence emission collection and tightly focused laser excitation beams. Few

investigators have reported limits of detection below the zeptomole range. Sub zeptomole detection limits have been achieved using CE by investigators including Soper et al. 38 (0.4 zmol) and Dovichi et al. 33 (0.050 zmol). Impressive sensitivities as low as single-molecule detection have also been reported for molecules in a owing stream, demonstrating the possibilities of LIF when used with CE. 42,43 In order to attain these sub zeptomole levels, an off-column sheath-ow detection arrangement was generally utilized to reduce the amount of background scatter. Neat dye solutions were used under conditions that optimized the quantum yield of the dye (), not necessarily desirable for real CE applications. High laser powers, time gating and data smoothing have also been utilized to achieve these results. The data presented here are for an NIR labeling dye used under 100% aqueous conditions in a fully automated instrument, suitable for routine use. Additional improvements in sensitivity could be achieved by increasing laser excitation power, improving the dye uorescence quantum yield, and optimizing the dye emission prole with the band-pass lters. The NIRLIF system uses more compact, rugged, and less expensive components with an expected lifetime of >100 000 h. The fully automated injection, separation, and data collection capabilities of the commercial CE system were preserved. 28 The fully automated system developed was then used to evaluate the suitability of the NIRD NN382 (40) as a peptide labeling agent. Six angiotensin-I (Ang-I) variants were selected as model peptides for derivatization and separation studies. 44 The calculated charge-to-mass ratios of the derivatives and excess dye (at pH 7.2) are given in Table 7, listed in expected order of elution in a normal polarity CZE separation. The smallest chargeto-mass ratio difference is between the human and Val-5 forms, where the substitution of valine for isoleucine

16
Table 7 Sequence of labeled Ang-I peptides listed in order of
decreasing charge to mass ratio and expected elution order in a normal polarity CZE system (amino acid differences from the human form are bold and underlined) Ang-I variant Goosesh Elasmobranch Salmon Human Val-5 human Bullfrog NN382 Sequence (NN382)-Asn-Arg-Val-TyrVal-His-Pro-Phe-His-Leu (NN382)-Asn-Arg-Pro-TyrIle-His-Pro-Phe-Gln-Leu (NN382)-Asn-Arg-Val-TyrVal-His-Pro-Phe-Asn-Leu (NN382)-Asp-Arg-Val-TyrIle-His-Pro-Phe-His-Leu (NN382)-Asp-Arg-Val-TyrVal-His-Pro-Phe-His-Leu (NN382)-Asp-Arg-Val-TyrIle-His-Pro-Phe-Asn-Leu Charge/mass 0.001237 0.001274 0.001289 0.001664 0.001674 0.001732 0.003006

ELECTRONIC ABSORPTION AND LUMINESCENCE

represents a difference of a single methyl group for the 2295 (formula weight) labeled peptide. The closely related decapeptides were labeled with the NIRD, separated using CE, and detected by NIRLIF. Derivatization of the peptides was achieved under aqueous conditions using 2.5 500 pmol of Ang-I in a 50-L sample (5 10 8 1 10 5 M). The uorescence response was linear over a 200-fold range (r > 0.9986) and the SNR of a 1.3-amol injection of the Ang-I variants was >15, indicating a limit of detection (SNR D 3) of <240 zmol. Four of six peptides were resolved from each other and excess dye using CZE. Two pairs of co-eluting peptides were successfully resolved using MEKC. Two neutral NIRDs were identied as suitable markers for measuring electroosmotic and micellar ow. 44 Another application evaluated with the NIRLIF system was in CGE. The suitability of NIRLIF detection with CGE was investigated. The purity of NIR-labeled primers was assessed, and detection linearity and sensitivity using CGE were evaluated. Slab gel electrophoresis has been used for many years for the analysis of proteins, oligonucleotides, DNA, and other biomolecules. The applications of this technique are very powerful and widespread, but also slow, labor intensive and prone to poor reproducibility. 45 CGE was developed to automate and simplify traditional gel electrophoresis methods. The advantages of using CGE include the ability to automate the process fully, provide on-line quantitative detection, and use smaller sample amounts. 30 32,45 The capillaries are superior at dissipating heat, allowing the use of higher voltages, resulting in improved efciencies and shorter run times. LIF detection has been successfully applied to DNA sequencing methods. Swerdlow et al. 46 reported

sequencing rates of up to 1000 bases per hour using uorescent-labeled primers. Another scheme uses uorescent intercalating dyes, such as those marketed by Beckman, for the analysis of ds DNA with LIF detection. Although the total sample throughput is still limited using CE compared with slab gel methods, this limitation is likely to be overcome. Through the use of one-channel, four-dye detection schemes and the development of multicapillary instruments, this limitation can be overcome. Beckman has recently introduced an eight-capillary CE instrument for DNA analysis and Ueno and Yeung reported on the use of a 100 capillary bundle. 47 CGE with LIF detection was utilized to assess the purity of NIR-labeled oligonucleotides. The CGE method showed great sensitivity, with limits of detection around 90 zmol and a 1000-fold linear dynamic range. The number of theoretical plates obtained for the primers exceeded 300 000. The excellent sensitivity and high separation efciency observed for the NIRlabeled primers indicate their suitability for many other CGE applications. 28 Sensitivity results are comparable to those reported by Williams and Soper 40 for a similar NIR-labeled primer. The excellent sensitivity and separation efciencies obtained for the NIR-labeled primers suggest they may be suitable for many of the other CGE applications described earlier. The use of uorescent-labeled primers in binding studies of synthetic oligonucleotides, such as antisense therapeutics, has been demonstrated. Vilenchik et al. 48 monitored the hybridization of a phosphorothioate target with a complementary labeled DNA probe. Protein to DNA binding studies were shown by Xian et al. 49 using a CE mobility shift assay with CGE. An assay to determine the activity of a transcription factor (SpP3A2), obtained from the nuclei of a single sea urchin egg, was based on the difference in mobility of a free or bound uorescent labeled DNA probe. 4.2.2 Application of Near-infrared Fluorescence in Immunoassays IA is a method of analysis that relies on specic interactions between an Ab and an antigen (Ag) to measure a variety of substances, ranging from complex viruses and microorganisms to simple pesticide molecules and industrial pollutants. To observe and measure this reaction, a label is introduced via a second Ab. Conventionally, this label consists of a radioactive isotope (radioimmunoasay (RIA)), an enzyme (enzyme-linked immunosorbent assay (ELISA)) or a uorescent molecule (uorescence immunoassay (FIA)). The use of Abs as analytical regents was rst reported in 1959 when Berson and Yalow successfully demonstrated the measurement of picogram levels of human insulin in samples of body

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

17

uids by RIA. 50 Since then, various IAs for detecting hundreds of molecules of endogenous and exogenous origin have been described. This technique proved to be reliable, fast, and very sensitive; many other RIAs have been developed for clinical and medical tests since then. The use of immunochemical techniques in the environmental eld was rst proposed in 1971 by Ercegovich, 51 who suggested the use of immunological screening methods for the rapid detection of pesticide residues and for conrming results of conventional analysis. An RIA for the insecticides aldrin and dieldrin was the rst reported IA for an environmental contaminant. 52 Although a few RIAs still exist in the medical eld, they are seldom used in environmental and food analysis because of the need for special handling and disposal of the radioactive materials. Radiolabels were gradually replaced with enzyme labels owing to the hazards associated with radioactive materials. ELISA, which was rst introduced by Engvall and Perlman in 1971, 53 has become perhaps the most popular IA format used in laboratories today. The modern diagnosis of many diseases, especially infectious diseases, is almost completely dependent on these assays. In diseases of global importance such as acquired immunodeciency syndrome (AIDS), 54 cysticercosis, 55 malaria, 56 lariasis, and schistosomiasis, 57,58 IAs play a key role in screening and diagnosis. Utilizing the advantages of uorescence over absorbance, conventional uorophores can be theoretically capable of detecting fewer than 106 molecules L 1 59 in conjugation with the highly specic activity provided by IAs. However, in practice, the high background from the biological samples autouorescence, or sample reagents and scattering and quenching effects of solvents, limit the detection to about 10 10 molecules L 1 . LIF provides an alternative to improve the sensitivity of uorescence in IAs. The use of LIF in IAs has been reviewed. 60 62 The limitations of conventional lasers as excitation sources are their high price, size, maintenance costs and their limited wavelength selection. LIF in the NIR region (600 1100 nm) offers several advantages. Recent advances in semiconductor laser technology have made the use of lasers more practical. The widespread use of NIR-emitting laser diodes in the telecommunications industry has made them more readily available. This type of laser is inexpensive (typically <$150) and small (1 cm) and has a much longer operating lifetime (>100 000 h). 63 The GaAlAs laser diode has drawn much interest because its emission wavelength of approximately 800 nm is compatible with several classes of polymethine cyanine dyes, which exhibit NIR uorescence. Detection in the NIR region allows one to replace commonly used photodiodes by APD. The APDs have excellent quantum efciency in the NIR region. Some of the advantages of APDs are that they are much cheaper, more compact, and

longer lasting. In addition, they have low internal noise and very low power consumption. All these features make the NIR uorescence IA highly amenable to miniaturization and can aid in the development of a portable, compact and rugged instrument for eld application. Heptamethine cyanine dyes are a class of NIR uorophores that have been used for DNA sequencing, pH and hydrophobicity determination, metal ion detection, and Ab labeling. 63 They are ideal for labeling Abs involving conjugation chemistry, e.g. the reaction of the isothiocyanato group (NCS) on the NIRD with the primary amine groups on Abs. These dyes have high molar absorptivities (ca. 105 L mol 1 cm 1 ), high quantum yields (20 40%), relatively short uorescence lifetimes (500 1000 ps) and are small (1000 Da). The small size of these dyes in comparison with the target molecule allows for a high number of labels per Ab without compromising Ag/Ab interactions. This implies higher selectivity and signal coupled with low background interference, in addition to the previously discussed advantages offered by the NIR region. Additionally, the use of a solid matrix generates a stronger signal by concentrating the uorescent molecules and thereby reducing quenching effects of the solvent. 2 Boyer et al. were the rst to demonstrate the feasibility using diode laser detection in the NIR region. 64 They compared the efcacy of the NIR assay method with the conventional ELISA method. Williams 65 constructed an NIR uorescence detector for detecting uorescence in an IA format. The set-up developed is illustrated in Figure 8. The instrumentation comprised an excitation source coupled with a ber-optic cable, a silicon photodiode as detector, a sample-holding apparatus that could be adapted to hold various forms of solid support matrices coupled with a motor drive, and a data acquisition interface. The instrument could detect concentrations of 100 pmol of NIRDs. Using this set-up, the near-infrared

Optical fiber

Motor drive Detector

Power supply

Power supply

Amplifier

A/D board

Laser

Power supply

Figure 8 Block diagram of NIR uorescence detector.

18
uorescence immunoassay (NIRFIA) on a nitrocellulose matrix was capable of detecting 5 10 10 M human immunoglobulin G (IgG). The overall assay could be performed in less than 2.5 h in the absence of the substrate development step which is employed in the conventional ELISA method. This NIRFIA allowed the detection of approximately 470 00 labeled Ab molecules. This method developed, however, had its disadvantages. A high degree of scatter generated by the membrane, problems with nonspecic binding of the conjugate, and its lack of compatibility with the most common format of modern IAs, the microtiter plate assay format, limit its practical use. In continuation of the development of solid-phase NIRFIA, the abovementioned issues and others were addressed in a study by Swamy. 2 An NIRFIA was developed based on the heptamethine cyanine dye NN382 (40). The dye used in this study had a high molar absorptivity (e D 180 000 L mol 1 cm 1 ) and quantum yield ( D 0.59, for dye Ab conjugate). The isothiocyanate functionality reacts selectively with the amino group of the Abs to form a stable thiourea bond. The presence of the sulfonated groups makes the dye highly water soluble, making it ideal for labeling Abs. Another advantage of the negatively charged sulfonate groups on the dye is that it minimizes nonspecic binding to the solid matrix (polystyrene (PS)). A solid-phase NIRFIA was systematically developed in the following steps. A LI-COR 4200 prototype uorescence microscope 41 was coupled with an orthogonal scanner and was evaluated for optimum depth of view. Next, the optimum scanner operating conditions were determined in terms of gain, offset, and scan speeds to optimize settings for maximum dynamic range and SNR and ensure reproducible scans. Appropriate modications to the mounting of the scanner microscope and
Thermoelectric cooler

ELECTRONIC ABSORPTION AND LUMINESCENCE

scanning platform were made to allow the examination of microtiter plates (Figure 9). The linear response of the detector was veried with increasing gain for a given voltage setting. A number of microtiter plates (solid matrix) were then evaluated in order to provide the one with minimum background noise. Once the reader had been congured and the solid support had been augmented, the procedure for covalent conjugation of NIRD NN 382 (40) to goat antihuman immunoglobulin G (GAHG) was systematically optimized in terms of pH, temperature, time of reaction, and reactant molar ratio. The conditions that provided the maximum results were a pH of 10, an initial dye : Ab ratio of 1 : 125, a temperature of 25 C and a coupling time of 1 h. These conditions are all critical parameters since any deviation results in underor overlabeling of the Ab, which, in turn, results in a low specic activity and detection ability. The absolute concentration of reactants was found to be critical for the rate of the coupling reactions; therefore, in the optimization procedure, stock solutions of dye (10 mg mL 1 ) and Ab (1 3 mg mL 1 ) concentrations were used. Although the conditions for coupling other species of Ab were not optimized, other conjugates were successfully prepared with high specic activity. The optimum conjugation procedure was dened by the conditions that produced an NIRD-labeled Ab with the highest specic activity in an immunosorbent assay for normal human immunoglobulin G (NHIgG). The best conjugate was used to determine detection limits for the assay and was able to detect 2 10 11 M of NHIgG. This provided a sensitivity about 10 times greater than that achieved by conventional labels in similar assays. Finally, the assay was validated by 100% agreement with clinically diagnosed samples for schistosomiasis and its cross-reactors. 2

Avalanche photodiode Focusing lens Laser diode Detector filter stack Collection lens ELISA plate Motor drive

IR analyzer 4000X

Figure 9 Model assembly to scan microtiter plates.

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

19

One major disadvantage of this method is the lack of commercial instrumentation for the assay. Quantication of the signal obtained in an image format is problematic. However, commercial software for quantifying on line signals from DNA sequences in the NIR region is available (LI-COR). Adaptation of similar software for microtiter plate scans would be immensely helpful. The method developed does have many advantages over conventional methods and the versatile nature of the scan bed does not limit this assay to the microtiter format. Technically, the scanner could scan any matrix, limited only by the physical dimensions of the scanner (ca. 23 cm and 61 cm long). This could allow for scanning multiple assays and 500 1000-well plates. The assay is very quick, requiring less than 20 min for the assay itself, and requires fewer steps than ELISA. The ability to detect lower concentrations of Abs would help in earlier diagnosis and an opportunity to test drugs against opportunistic infectious agents at an earlier stage. The labeling of Abs with NIRDs provides an important advancement to IAs and the comparatively small size of the NIRD allows for a higher molar ratio of NIRD per Ab. This in turn yields a higher signal and allows for lower detection limits. The assay has also been validated for clinical samples and could be further evaluated in the development of a diagnostic test. In principle, various bioanalytical methods can be developed using the NIR immunochemistry described in the preceding section. To demonstrate the versatility of the method, Swamy et al. developed two bioanalytical applications utilizing NIRFIA in collaboration with the State University of New York (SUNY) at Brooklyn and the Department of Entomology and Environment Toxicology at the University of California, Davis, CA, USA. The rst application involved an NIR-labeled Ab in the detection of extracellular Ag expressed on cells. This demonstrated the feasibility of using NIR-labeled Abs in clinical imaging applications such as NIR optical tomography. The second application uses the NIRFIA as an analytical tool in environmental applications in the quantitative analysis of two pesticides, bromacil and fenvalerate. In the evaluation of the NIRD-labeled Ab for imaging applications, the NIRD NN82 was coupled to monoclonal Ab E48 (MAb E48) and the conjugate was evaluated in a direct assay to detect a 22-kD Ag expressed on the surface of human squamous cell carcinoma line (HuSCC) A431. The specicity of binding was conrmed in a competitive assay. The preliminary results showed good sensitivity and specicity of the NIRD-labeled Ab. In the environmental application, the assay was evaluated in a rapid tracer format competitive assay for the detection of the pesticides bromacil and pyrethroid. In this assay, the specic Ab captured on the surface of microtiter plate

well and the tracer with uorescent label compete with the analyte for the Ab. The results obtained showed that the NIRFIA was at least as sensitive as ELISA, with both assays detecting pesticides in the micrograms per liter (parts per billion) range. 2 The chemistry and the instrumentation of the NIRFIA technique for environmental applications have been developed, but are amenable to further miniaturization. Further optimizations would allow for the future construction of compact instrumentation for analysis which could conduct fast and reliable assays at remote locations. In continuation of this approach, preliminary results obtained in our laboratory demonstrated the feasibility of using the NIRFIA approach for an NIR/ber optic immunosensor (FFOI). This approach is particularly useful for measuring small amounts of analyte and can be automated and carried out in remote locations. Several uorescent immunosensors have been reported; however, lack of commercial instrumentation and labels limit the use of these techniques and most applications utilize UV/VIS dyes. The applications of the visible uorescent immunosensors described are all susceptible to interference from biomolecules such as bilirubin and porphyrins. A comprehensive review of immunosensors has been published by Robinson et al. 66 Danesvar 67 developed a NIR/FFOI for detection of trace amounts of human IgG and Legionella pneumophilia. They optimized the assay on different solid phases such as poly(methyl methacrylate) (PMMA) and PS. The PS coating method eliminated the activation process required with PMMA, thereby reducing the preparation time by 18 h; in addition, it also eliminated the protein G step of the previous assay. The assay was carried out in a sandwich format (Figure 10). The set-up used to measure the signal (Figure 11) allowed concentrations of 10 11 M for IgG (SD D 0.13) and 0.5 ng mL 1 for Legionella pneumophilia sera type 1 (LPS1) 67 to be detected. These results were comparable to those attained by conventional ELISA. Another application using a similar format was developed by Evans 68 and provided a competitive assay for the pesticide bromacil. A detection limit of 5 ppb was obtained, which was slightly higher than that reported for ELISA (0.1 ppb). However, the method had advantages since it did not require extensive sample preparation, the assay time was substantially reduced, and the assay could be adapted for remote site analysis. 68 The NIR/FFOI technique in principle is a exible methodology, which can be theoretically adapted to develop assays for any compound for which Abs are available, including infectious agents, serum analytes, and environmental pollutants. This technique provides a real-time analysis with automated readout capabilities, eliminating the need for operator intervention or

20

ELECTRONIC ABSORPTION AND LUMINESCENCE

,, ,, ,, ,, ,, ,, ,, ,, ,, ,,

,, ,, ,, ,, ,, ,, ,, ,, ,, ,,

,, ,, ,, ,, ,, ,, ,, ,, ,, ,,
D

,, ,, ,, ,, ,, ,, ,, ,, ,, ,,
G G

Antibody Antigen Conjugate D

Figure 10 Sandwich assay on ber-optic probe. One of the rst NIR uorescence-based HPLC detectors was developed by Ishibashis group. They detected 0.3 pg of a carbocyanine dye, 70 with instrumentation comprising a 3-mW diode laser emitting at 780 nm with a PMT cooled to 20 C for detection. They reported a detection limit of 1.9 pg at room temperature. Sternberg et al. 71 of Beckman Instruments used a 2.5-mW diode laser coupled with a commercial diode-array detector and achieved a detection limit of approximately 10 10 M for a carboxyl cyanine-labeled oligonucleotide. Winefordner et al. explored the detection of cyanine dyes in different instrumental congurations, 72 using a diode laser for excitation with a PMT for detection. They achieved detection of 46 000 molecules in a liquid jet uorescence spectrometer. Karnes et al. 73 used diode laser-induced NIR uorescence for detection of ICG in plasma. They demonstrated a detectability of more than two orders of magnitude over absorbance. Another NIR HPLC application was reported by Kuklenyik, 74 who developed and compared two detectors for NIR uorescence detection based on a
780 nm Interference filter

Lock-in amplifier Laser diode 780 nm Excitation Photodiode Emission Fiber sensitive terminal Digital voltmeter

Figure 11 Fiber-optic immunosensor set-up. complicated sample preparation. Although the detection optics for the assay were originally constructed on a 2 3 ft optical table, the set-up can be reduced in size. The preliminary sensitivity, selectivity, and simplicity of the assay are encouraging and further design renement and other applications should aid the development of versatile NIR/FFOI. 4.2.3 Application of Near-infrared Fluorescence in High-performance Liquid Chromatography Another bioanalytical application developed using the advantages of NIR uorescence is HPLC detectors. In recent years, the ability to measure ultralow levels of pharmaceuticals in biological matrices has presented signicant challenges. Using uorescence as a tool for detection, sensitivities of picograms per milliliter have been achieved. Rahavendran and Karnes have described the advantages of LIF detection in HPLC. 69 There have not been many publications dealing directly with HPLC detection in the NIR region. Until recently, two groups worked on the development of applications of NIR uorescence in HPLC, Winefordners group at the University of Florida and Ishibashis group in Fukuoka, Japan.

Laser diode

Flow-through cell

Lenses 830 nm Interference filter or filter bank

Photodiode

Figure 12 Optical path of NIR/HPLC uorescence detector.

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS


0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0.5 1 1.5 2 2.5

21

HPLC

Waste

Pump Flow-through cell

Laser source 20 mW Serial link

Signal (V)

Eluent (MeOH) volume (mL)


Figure 14 Detection of three consecutive injections of IRD40
12345 Detector Microcomputer

on a C18 column.

Amplifier

Digital voltmeter

Figure 13 Experimental set-up of NIR/HPLC uorescence


detector.
NCS

O N
+N

NaO3S

SO3Na

(41)

runs with C18 HPLC columns. Detector signals for three consecutive injections of IRD40 at 10 10 M concentration are shown in Figure 14. Detection limits of 1.22 10 11 and 5.89 10 12 M were achieved for the two dyes and the numbers of dye molecules detected were calculated to be 36 500 and 17 600, respectively. 74 The overall results showed that the less expensive, more efcient photodiode was sensitive for detection in the NIR region. The detector was designed to collect signals at 820 nm, and a better match of the dye emission with the detector and better light collection efciency should help improve the sensitivity much more. Even in the early stage of development in this direction, the NIR uorescence method shows sensitivity comparable to or better than those of conventional methods. Further evaluations and modications could help achieve much greater sensitivities. 4.3 Environmental Applications of Near-infrared Fluorescence The fundamental principle involving the analytical applications of dyes to study the microenvironment essentially involves the changes induced by the analyte that can be directly measured by spectral changes. These changes depend on the concentration of the analyte and the interaction between the analyte of interest and the dye. Typically these probes involve a cation-selective receptor either as an integral part of the chromophores p-system or covalently attached to the uorophore via an alkyl tether. 75 77 The coordination of the cation affects the spectral position of absorption and emission bands, molar absorptivity, and, to a smaller extent, the uorescence quantum yield. The interaction of a metal ion with an organic reagent might result in the enhancement of the uorescence or the quenching of the uorescence of the organic reagent in the presence of an analyte. A statistical study showed that about 89% of luminescent methods are based on the enhancement of uorescence and about 11% are based on quenching reactions.

+N

ClO4

(42)

silicone photodiode and an APD. The merits of this type of detector were also compared with those of conventional PMT detectors. An HPLC system (SSI gradient system) with a high-pressure pump capable of providing 500 psi pressure controlled by an IBM computer was coupled with an in-house developed excitation and detection system. The experimental set-up of the excitation and detection and the nal set-up are shown in Figures 12 and 13. It comprised a silicon photodiode detector coupled with a 24-L HPLC ow cell shown in Figure 12 and, in the set-up with an APD, a modied Model 4200 LI-COR microscope 41 was used instead of the silicon photodiode. Detector performance was evaluated with two NIRDs, IRD40 (41) and 1,10 ,3,3,30 ,30 -hexamethyl4,40 ,5,50 -dibenzoindotricarbocyanine perchlorate (42), rst without an HPLC column followed by subsequent

22
The investigation of toxic metal ions in the environment has been of growing interest in the past few decades and the decreasing permissible limits for most of these metal ions set by major health organizations such as the World Health Organization (WHO) and the United States Environmental Protection Agency (USEPA) have provided a tremendous thrust for the development of sensitive analytical techniques. Most of the publications devoted to the detection of metal ions by means of spectroscopic techniques used UV/VIS probe molecules such as rhodamine, uorescein, 8-hydroxyquinoline, and their derivatives. 78 83 Table 8 provides a list of some of the different uorescent sensors that have been used for the determination of the most commonly studied metal ions, namely Al(III), Be(II), Co(II), Fe(III), and LiC . Unfortunately, spectral interference is signicant in this region. The use of NIRDs is a better alternative. 84 90 The low background interference observed for other molecules in the NIR spectral region together with the longer Raman shift offers better SNRs. Casay et al. 91 investigated tetrasubstituted NPc NIRDs as potential probes for the determination of toxic metal ions. They developed the rst NIR optical probe that could detect lead, lithium, and cadmium at parts per billion levels 91 and potassium at 0.566 ppm. 92 More recently, Tarazi 93 reported the detection of metal ions using three novel

ELECTRONIC ABSORPTION AND LUMINESCENCE

NIRDs, namely TG 170 (43), NN525 (44), and JCM15C5 (45). The spectral characteristics of the three dyes are listed in Tables 9 11. They evaluated TG 170 for the detection of Al(III) and Be(II), NN525 for the detection of Fe(III) and Co(II) ions, and JCM-15C5 for the detection of LiC in the presence of other interfering ions.
HO CO2H Cl N
+N

HO2C

OH

CF3CO2

(43)

O N
2+

O HO2C SO3

(44)

Table 8 Different uorescent methods currently employed for detection of various metal ions
Metal ion Al(III) Reagent Alizarin Red S Lumogallion 8-Hydroxyquinoline Morin N-Salicylidene-2-hydroxy-5-sulfoaniline Superchrome Carnet Y 2-Hydroxy-3-naphthoic acid 2-Ethyl-5-hydroxy-7-methoxyisoavone 2-Hydroxy-3-naphthoic acid Morin Al(III) Pontachrome BBC Al(III) Superchrome Blue Black 2-[2-Hydroxy-1-naphthyl]dithiocarbazic acid-4-chlorobenzyl ester 1-(2-Pyridylazo)-2-naphthol Al(III) Pontachrome BBR 40 -(4-Methoxyphenyl)-2,20 ,200 -terpyridyl Rhodamine B 2,20 ,200 -Terpyridyl Dibenzothiazolylmethane 5,7-Dibromo-8-hydroxyquinoline 1,4-Dihydroanthraquinone 1,5-Dihydroanthraquinone 1,8-Dihydroanthraquinone 8-Hydroxyquinoline Methoda E E E E E E E E E E E Q Q E Q Q Q Q E E E E E E Detection limit (ppm) 8 10 2 3 10 2 3.2 10 1 5 10 3 2.5 10 4 5 10 3 8 10 5 8 10 2 8 10 4 1.6 10 1 1.8 10 4 1.8 10 4 10 4 1.2 10 2 1.8 10 4 1.8 10 4 10 4 1.6 10 3 10 3 10 3 2 10 2 10 1 1.1 5.9 10 2 2 10 2 2 10 10 2 10 1 1 10 2 5 10 1 5 10 2 2.5 10 1 2.5 5 10 2 5 10 5 10 2 5 10 5 10 2 7 10 4 10 2 1
1

Be(II)

Co(II)

Fe(III) LiC

1 1 1

E D enhancement of uorescence; Q D quenching of uorescence.

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

23
3500 3000 2500 2000 1500 1000 500 0 6 9 12 15

O O

O O O

NH N I
+N

(45)

Fluorescence intensity (a.u.)

Table 9 Spectroscopic characteristics of TG170 in various


solvents Solvent Dimethyl sulfoxide Methanol Buffer (pH 5.9) lmax (nm) 819 805 791 lem (nm) 822 812 803 f (%) 0.167 1.30 2.48 Log e (L mol 1 cm 1 ) 4.96 5.40 5.99 NN525.

Concentration of Fe(III) (10

M)

Figure 15 Calibration curve for detection of Fe(III) with

Fluorescence intensity (a.u.)

1000 900 800 700 600 500 400 1.5 1.67 1.88 2.15 2.51 3 3.75 5 7.5

Table 10 Spectroscopic characteristics of NN525 in various


solvents Solvent Acetonitrile Dimethylacetamide Dimethylformamide Dimethyl sulfoxide Methanol Tetrahydrofuran Water lmax (nm) 664 672 672 676 663 669 661 lem (nm) 673 681 681 688 670 676 666 f (%) 0.86 0.92 0.81 0.73 0.87 0.91 0.03 Log e (L mol 1 cm 1 ) 5.64 5.74 4.67 4.69 5.72 5.76 5.38

Concentration of Co(II) (10 8 M)


Figure 16 Calibration curve for detection of Co(II) with
NN525.

Table 11 Spectroscopic characteristics of KVA22 in various


solvents Solvent Acetone Acetonitrile Dimethyl sulfoxide Methanol Methylene chloride lmax (nm) 780 776 793 777 787 lem (nm) 799 799 809 780 803 f (%) 3.02 1.58 2.03 1.52 2.74 Log e (L mol 1 cm 1 ) 4.12 4.83 4.66 4.81 4.96

The hydroxy carboxy functionality of TG 170 (43) has a known selective complexation with Al(II) and Be(II) ions. Using this dye, they reported a detection limit of 52 ppb for Al(III) and 24.6 ppb for Be(II) ion. For the NIRD NN525 (44), only Fe(II), Fe(III), and Co(II) ions resulted in quenching of uorescence. Detection limits of 3.54 ppb for Fe(III) and 1.55 ppb for Co(II) were obtained. The calibration curves obtained for the

two metal ions are illustrated in Figures 15 and 16. The crown ether functionalized cyanine dye JCM-15C5 (45) was used for detection of LiC ions in the presence of other interfering ions. They obtained a linear plot of uorescence intensity for LiC amounts ranging from 0.0347 to 0.222 ppb (Figure 17). A detection limit of 0.0743 ppb was reported. The stability constant for the JCM-15C5 complex was higher than those achieved in other studies. The method development represents a signicant improvement over conventional methods. Owing to the minimal interferences encountered in the NIR region, it eliminates the need for extensive sample preparation, and additionally it allows for the development of tests that can be carried out at remote sites by rugged compact instrumentation. Another novel application developed with NIRDs for the detection of Ca2C was reported by Akkaya and Turkyilmaz. 94 They developed a squaraine-based uorescent sensor for calcium [Scheme 6, (47)]. The probe developed was very sensitive to Ca2C concentrations in the

24
Fluorescence intensity (a.u.)
450 430 410 390 370 350

ELECTRONIC ABSORPTION AND LUMINESCENCE

12

16

20

24

28

32

Concentration of Li (109 M)
Figure 17 Calibration curve for detection of LiC with
JCM-15C5 (45).
CH3 HO O t-BuO2C N CO2t-Bu O N CO2t-Bu CO2t-Bu

(46)
i, ii

CO2t-Bu N t-BuO2C O CH3

use of an inexpensive commercial laser diode emitting at 690 nm for excitation and a photodiode for detection, opening avenues for the development of compact, rugged systems free from background interferences for cellular ion-ux studies and real-time imaging applications. 94 Another unique environmental application developed was an NIR phase-resolved uorescence spectroscopybased ber-optic corrosion sensor for the detection of aluminum and iron and their corrosion by-products by Chin et al. 95 The method essentially involves an NIRD that is embedded in a ber-optic cladding, coupled with appropriate excitation and detectors and integrated with an in-house phase-resolved uorescence spectroscopy board. A block diagram of the set-up is shown in Figure 19. They studied three in-house synthesized NIRDs, TG1 (48), TG2 (49), and TG3 (50) for complexing Al(III) ions. The method was more effective than conventional uorescence techniques because it measures the lifetime changes of the dye which are independent of the surrounding environment as opposed to uorescence. Table 12 lists the uorescence lifetimes for the three dyes in the presence of Al(III) ions. This method developed was very versatile as it could be integrated into airframe and wings of aircraft or other critical failure locations (fasteners, bolts, composite joints, welds) for measuring corrosion. Thereby, strain and structural fatigue can be monitored using NIR technology. The preliminary results obtained were very encouraging and future potential uses of this system could include monitoring composite life cycles and corrosion and structural fatigue of launch pads, rockets, space structures, and advanced aircraft. 96,97 4.4 Other Applications of Near-infrared Fluorescence

O t-BuO2C N

O HO t-BuO2C
2+

N CO2t-Bu O

CO2t-Bu OH O

In this section, we describe other analytical applications developed with NIR uorescence. The rst application discussed is the determination of hydrogen ion concentration (pH), which is an important part of many
60 a b c d 30 e 15 0 690 f

CO2t-Bu H3C N t-BuO2C

Emission intensity

45

(47)

Scheme 6 Synthesis of Ca2C uorescent chemosensor [Akkaya


and Turkyilmaz 94 ].

740

790

840

micromolar range (Figure 18). Signal changes resulting from the loss of the donor alkylamine functions on Ca2C chelation, decreasing the molar absorptivity from 300 000 to 50 000. The lmax of the dye at 698 nm permits the

Wavelength (nm)
Figure 18 Emission spectrum of uorescent chemosensor as a
function of Ca2C concentration.

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS


Reference filter (785 5 nm)

25

785 5 nm < 850 nm

Sensing region

, ,,,,, ,

Diode laser (785 nm)

,,,,,,,,,,,, ,, ,,

APD

Optics

Fiber coupler

Fiber support SPECs proprietary digital RF mixing electronics

Fiber coupler

>805 nm Optics

Figure 19 Fiber-optic set-up for phase-modulated uorescence detection of metal ions.


CO2H OH O N I
+

Table 12 Lifetime data for NIRDs TG1, TG2 and TG3


Dye
N

Dye concentration (M) 3.71 10 3.71 10 2.33 10 2.33 10 6.59 10 6.59 10


6 6 6 6 6 6

Al(III) concentration (M) 0.00 6.32 10 0.00 6.32 10 0.00 6.32 10


3

t (ns) 0.794 0.523 0.792 0.561 0.387 0.455

(48)

CO2H OH

TG1 TG1 TG2 TG2 TG3 TG3

O N I
+

(49)

OH CO2H

O N I
+

(50)

analytical procedures. The use of uorescent dyes and indicators for pH measurement is well documented in the

literature. A number of indicators with spectral characteristics in the UV/VIS region have been reported, 98 101 but only a few have been reported for pH measurements in the NIR region. Several classes of NIR pH-sensitive dyes have been developed in our laboratories at Georgia State University in collaboration with Strekowski et al. 102 105 Of these pH-sensitive dyes, the most useful include the bis(aminodiene)ones that undergo transformation from the keto form to the cyanine enol form. A pH-sensitive dye (51) (Scheme 7) showed distinct bands under acidic and basic conditions (Figure 20). The absorbance maximum at 531 nm undergoes a strong bathochromic shift in acidic conditions with a concomitant change in uorescence spectra with an absorbance band at 709 nm. The change in absorbance maximum is attributed to protonation of the oxygen atom which induces a shift to the cationic enol form of the dye and restores the cyanine chromophore to give a bathochromatic shift to the NIR region. Another pH-sensitive NIRD (53) was evaluated. Representative spectra of the NIRD trapped in Naon

26
O N

ELECTRONIC ABSORPTION AND LUMINESCENCE

pH 12.11

pH 12.82

pH 12.70

pH 12.88

pH 11.07

Absorbance ( 10 1)

1.00 0.80 0.60 0.40 0.20 0.00 380 480 580 680 780 880

(51)
+OH +H+

OH N
+

Wavelength (nm)
Figure 21 Absorption spectra of NIRD (53) entrapped in
Naon at different pH.
Cl Cl

(52)

Scheme 7 Keto-enol transformation of pH-sensitive NIRD


(51).

Cl 1.4 1.2 1.0

N n-Bu

EtO

N n-Bu

Absorbance

(54)
0.8 A 0.6 Cl 0.4 Cl 0.2 0.0 300 400 500 600 700 800 900 N n-Bu N+ n-Bu Cl B
+OEt +H+

(55)

Wavelength (nm)
Figure 20 Absorption spectrum of pH-sensitive NIRD (51) in
(A) acidic and (B) basic conditions.

Scheme 8 Novel pH-sensitive NIR ethoxy adduct of IR-1048


(54).

matrix at different pHs are shown in Figure 21. The changes in the spectra in the pH range 11 12.4 were reproducible. Owing to the extremely acidic environment of the Naon membrane, the dye provided a useful pH range, which deviated from the pKa value of the dye alone. This study demonstrated the use of Naon as a substrate for immobilizing NIRDs and the feasibility of developing an NIR pH sensor.
HO2C Cl N Br
+N

CO2H

(53)

Mason et al. 105 recently prepared a number of C2derivatized indolenine NIR chromophores that exhibit unique spectral properties as a function of pH. Of this series, JCM-1048-OEt (54) (Scheme 8) was studied by Tarazi. 93 The molecular structure of the dye contains an ethoxy moiety covalently linked to the C2-position of the 3H-indole. This addition results in the interference of the cationic delocalization that is typical of cyanine dyes and produces a complex absorption spectrum with a lmax at 511 nm. However, addition of acid regenerates the cationic chromophore IR-1048 (55) to give a dramatic red shift to 1050 nm. The spectral characteristics of the dye in different pHs are shown in Figure 22. As can be seen, the dye exhibits two characteristic absorptions, one in the NIR and the other in the UV/VIS region. The dye exhibited a linear range for the NIR peak over the pH range 6 9 in acetone, pH 5 9 in acetonitrile and pH 6 12

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

27

1.20 1.00

0.80 0.60 0.40 0.20 0.00 400 600 800 1000

Wavelength (nm)
Figure 22 Absorption of JCM 1048-OEt (54) at different pHs.
Cl O N n-Bu n-Bu N Cl

(56)
+OH +H+

Cl OH N n-Bu n-Bu + N Cl

(57)

Scheme 9 Structure of pH-sensitive NIRD JCM 648/932


(56/57).

in water as the solvent. In addition to the C2-derivatized NIRD, Mason et al. 106 reported the synthesis of a bis(aminodiene)one, JCM-648/932 (56) (Scheme 9) that exhibits pH-dependent absorptions in the NIR region. In neutral or basic conditions the equilibrium favors the keto form with an absorbance maximum of 648 nm in methanol. However, addition of acid results in the protonation of the carbonyl to restore the cyanine system. This acidic species (57) shows electronic absorption at 932 nm. The transformation from the keto to the enol form is fully reversible and can be carried out repeatedly. Another application developed using NIR uorophores is uorescence detected circular dichroism (FDCD). This method is similar to absorption CD, but uorescence intensity as opposed to absorbance is the measure of molecular ellipticity. Absorption CD is a valuable tool for examining the molecular chirality of proteins in the ground state. However, owing to the limited

number of chiral centers, CD is often limited to qualitative measurements and global conformational changes instead of optical activity about specic centers. Tinoco and Turner reported the ability to circumvent conventional problems with CD measurements by the FDCD technique. 107,108 FDCD can be used to measure the CD of a single contributing chiral uorophore while avoiding nonuorescent chiral absorbers and achiral uorophores. 108,109 For example, uorescence from a single tryptophan residue has been measured in the midst of other chiral absorbing residues present in proteins. Structural studies of proteins with and without ligand binding have also been reported. 100 112 Similarly to uorescence spectroscopy, FDCD makes use of a 90 angle relative to the excitation source for detecting the uorescence. Unlike uorescence, where plane polarized light is used for excitation, circularly polarized light is used in FDCD. When in the absence of energy transfer and only a single chiral uorophore exists at the analytical wavelength, the detected FDCD is for a particular population of uorophores. This situation may be inconceivable in many applications where many proteins may be present in a mixture. In order to avoid complications with matrix interferences, chromatographic techniques have been used in conjunction with FDCD. Yeung et al. 113,114 reported the use of LIF with FDCD coupled to HPLC and CE. They reported detection limits of 170 and 0.07 pg for riboavin by HPLC/FDCD and CE/FDCD, respectively. Thomas et al. reported the use of multidimensional FDCD for resolving mixtures without physical separation. 111,112 This method utilized the differences in the excitation and emission bands for resolving the uorescence spectra of optically active uorophores. Resolving mixtures of optically active uorophores based on their uorescence lifetimes has also been reported. 115,116 Since CD is a weightedaverage measurement, 115 many chromophores present in biomolecules contribute to the CD signals. This presents a quantitative limitation in CD measurements. Although the same rules apply to FDCD, intrinsic uorescence from optically active uorophores limits the measured CD to a few possible molecules such as tryptophan, tyrosine, and phenylalanine in proteins. However, when chiral nonuorescent molecules absorb in the ultraviolet (UV) region, the radiation intensity incident on the uorophore of interest can be weakened. Consequently, the emission intensity may diminish. Considering the lower quantum yields associated with uorescent protein residues and the number of potential interfering transitions, CD and FDCD at shorter wavelengths are limited. Long-wavelength alternatives to UV CD measurements have been reported. Keiderling discussed the use of vibrational circular dichroism (VCD), a chiroptical

Absorbance

28
analog of IR spectroscopy. 117 This technique measures the intrinsic vibrational modes of proteins which avoids some of the limitations of conventional absorbance CD. Sophianopoulos et al. used extrinsic NIR chromophores as structural probes in near-infrared circular dichroism (NIRCD) measurements. 118 Since synthetic probes possessing various properties are readily available, photophysical limitations of intrinsic chromophores can be avoided. Although VCD and NIRCD have the advantage of avoiding biological background interferences, they lack the sensitivity gained in FDCD. Since there are very few proteins that exhibit long wavelength uorescence, greater sensitivity, selectivity, and spectral resolution can be gained through the use of near-infrared uorescence-detected circular dichroism (NIRFDCD) of extrinsic uorophores. Many biomolecules typically absorb only at lower wavelengths, and thereby complex samples can be potentially analyzed without physically separating matrix components. Typically, NIRDs are optically inactive, low molecular weight (MW 1000 g mol 1 ) symmetrical compounds that do not yield FDCD. When placed in an asymmetrically perturbed environment, as in protein binding sites, chirality may be induced, resulting in Cotton effects. 118 Therefore, these compounds must meet two criteria in order to exhibit FDCD: rst, the molecule must be uorescent in the NIR region, and second, it must possess induced chirality through noncovalent interactions with the macromolecule clearly indicating that binding has occurred. In our laboratories NIRFDCD was used in order to gain a better understanding of the structure of NIRDs that bind proteins. An understanding of dye binding to albumin may relay important information concerning these dyes as labels or probes in analytical techniques such as the separation of proteins with HPLC, gel permeation chromatography (GPC) or in gel electrophoresis. Meadows et al. 119 recently investigated the NIR squarylium dye NN525 (44) as a non-covalent probe for protein structural determinations. This dye, owing to its rigid squarate residue, has greater photostability relative to its cyanine counterpart. They found that it forms a stable complex with serum albumins with binding constant in excess of 106 L mol 1 compared with 105 L mol 1 for many other conventional dyes. 120,121 This in turn added to the ease of detection. Also, the high quantum yield and molar absorptivity permit the use of smaller amounts of dye relative to visible counterparts. Preliminary results showed that NIRFDCD of squarylium uorophores can be measured using 10 100-fold lower dye concentrations in comparison with normal absorbance CD. 119 The advantage of using lower concentrations of dyes simplies the assessment of competing equilibria associated with high concentrations such as dye or protein aggregation.
0.35 0.30

ELECTRONIC ABSORPTION AND LUMINESCENCE

NN525 / human NN525 / horse

Absorbance

0.25 0.20

NN525 / chicken 0.15 0.10 0.05 0.00 600 620 640 660 680 700

Wavelength (nm)
Figure 23 Absorption spectra of NN525 (43) bound to different proteins.
0 Chicken 10 Horse

20 30 Human 40 600 620 640 660 680 700

Wavelength (nm)
Figure 24 NIRFDCD spectra of NN525 (43) bound to different
proteins.

Dye aggregation may in some instances result in the absorption of circularly polarized light. 122 A common nding for many of the NIRDs previously studied is the enhancement of photophysical properties upon binding to hydrophobic environments (Figure 23). 118,123 125 An increase in these properties permits the use of even lower probe concentrations. This can be advantageous since lower ratios of bound uorophores can leave proteins virtually unperturbed structurally. Previous reports by Sophianopoulos et al. 118 support the binding of NIR squarylium compounds to similar, if not identical, binding sites on bovine serum albumin (BSA) with immeasurable conformational changes. Many of the symmetrical NIR compounds do not have measurable optical activity in the protein-unbound state. When these dyes bind in asymmetric binding sites of proteins, induced Cotton effects can occur. As can be seen in Figure 24, the relative FDCD intensities found for the three different albumin dye complexes is in accordance with the absorbance spectra. This indicates

NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS

29

that those transitions occur through asymmetric perturbations in the uorophore. Structural differences in the binding sites occupied by NN525 cause intensities to differ. Although not conrmed, previous data supported a common binding site near positively charged amino acid residues. 118 Homology in this site among different species may account for the common negative Cotton effects observed for different albumins. Differences in the hydrophobicities and/or electrostatic interactions at these sites may explain the intensity variations exhibited for the different species. Changes in the extent of the association or weak induced chirality can result from weaker noncovalent interactions. The squarylium dyes had large molar ellipticities (105 cm2 dmol 1 ) when bound to BSA. Although induced CD may occur with binding to macromolecules, optical activity due to intramolecular forces, such as dye aggregation 123,126 in aqueous solutions, is less likely at lower concentrations used in FDCD. In many cases these complexes quench uorescence, but absorb circularly polarized light. Therefore, the CD intensities may falsely indicate asymmetric interactions in the protein-bound dye. The lower concentrations (10 7 M) needed in these NIRFDCD studies help prevent solubility limitations that can cause the formation of aggregates, avoiding extraneous Cotton effects. Hence the probability of FDCD data containing artifacts from aggregates is small relative to CD. The use of extrinsic uorophores in NIRFDCD can provide an abundance of complementary information about conformational changes near the uorophore. The main mode of binding of NIR squarylium dyes to BSA was determined to be hydrophobic, although electrostatic forces may also contribute. Van der Waals contacts near the dye binding site may enhance their association to proteins. The use of CD in conjunction with tryptophan FDCD in proteins and NIRFDCD of noncovalently bound dyes can provide a more detailed description of protein structure and interactions of proteins with ligands. A further advantage of NIRFDCD, the need for lower concentrations, prevents the occurrence of probe and protein intramolecular interactions, which simplies data interpretation.

environmental systems where background interference is often a major concern. The availability of commercial NIRDs and NIR instrumentation, although limited, has provided a tremendous advance in the development of applications in this region. The commercial dyes or instrumentation can be tailored to specic applications and aid in interfaces with other commercial instrumentation readily available. The photostability of NIRDs is a cause for concern. However, recent studies have focused on the development of functionalized photostable NIRDs to overcome this problem. The combination of diode laser excitation sources with photodiodes for detection and coupling of these to suitable ber-optic interfaces aid in miniaturization and allow for development of remote sensing applications. Time-resolved measurements for discrimination between similar species have made the method very useful both in biological applications such as DNA sequencing, cytology, immunoasssays, and intracellular measurements and in environmental applications in determining metal ions, pH and microenvironment determination. Other areas that utilize the advantages include photodynamic therapy, thermal transfer printing, optical recording, transparent bar codes, and forgery prevention. Progress continues to be made with more applications being developed using the emerging novel NIR uorescence technology.

ABBREVIATIONS AND ACRONYMS


Ab ADC Ag AIDS Ang-I APD BSA CBQCA CD CE CGE CZE DRAW ELISA FDCD FFOI FIA FITC GaAlAs GAHG Antibody Analog-to-digital Convertor Antigen Acquired Immunodeciency Syndrome Angiotensin-I Avalanche Photodiode Bovine Serum Albumin 3-(4-carboxybenzoyl)2-quinolinecarboxaldehyde Circular Dichroism Capillary Electrophoresis Capillary Gel Electrophoresis Capillary Zone Electrophoresis Direct Read After Writing Enzyme-linked Immunosorbent Assay Fluorescence Detected Circular Dichroism Fiber Optic Immunosensor Fluorescence Immunoassay Fluorescein Isothiocyanate Gallium Aluminum Arsenide Goat Antihuman Immunoglobulin G

5 CONCLUSIONS
The discussions in this article demonstrate the various advantages of working with the novel emerging technology of NIR uorescence. Reduction in background interference offered by this method is the greatest advantage. No other analytical method known thus far can compete with the highest possible sensitivity achievable, i.e. single-molecule detection, offered by LIF. NIR detection provides advantages in complex biological and

30
GC GPC HDITCP Gas Chromatography Gel Permeation Chromatography 1,10 ,3,3,30 ,30 -Hexamethyl-4,40 ,5,50 dibenzo-2,20 -indotricarbocyanine Perchlorate 1,10 ,3,3,30 ,30 -Hexamethyl Dicarbocyanine Highest Occupied Molecular Orbital High-performance Liquid Chromatography Human Squamous Cell Carcinoma Line Immunoassay Indocyanine Green Isoelectric Focusing Immunoglobulin G Infrared Intersystem Crossing Isotachophoresis Laser-induced Fluorescence Legionella Pneumophilia Sera Type 1 Lowest Unoccupied Molecular Orbital Micellar Electrokinetic Chromatography Metal Ion Phthalocyanine Mass Spectrometry North Atlantic Treaty Organization Normal Human Immunoglobulin G Near-infrared Near-infrared Circular Dichroism Near-infrared Dye Near-infrared Fluorescence-detected Circular Dichroism Near-infrared Fluorescence Immunoassay Near-infrared Laser-induced Fluorescence Naphthalocyanine Phthalocyanine Polymerase Chain Reaction Poly(methyl methacrylate) Perturbational Molecular Orbital Photomultiplier Tube Pariser Parr Pople Molecular Orbital Polystyrene Radioimmunoasay Signal-to-noise Ratio Thin-layer Chromatography United States Environmental Protection Agency Ultraviolet Ultraviolet/Visible Vibrational Circular Dichroism World Health Organization Write Once Read Many

ELECTRONIC ABSORPTION AND LUMINESCENCE

RELATED ARTICLES
Biomedical Spectroscopy (Volume 1) Biomedical Spectroscopy: Introduction Fluorescence Spectroscopy In Vivo Biomolecules Analysis (Volume 1) Circular Dichroism in Analysis of Biomolecules Fluorescence-based Biosensors Clinical Chemistry (Volume 2) Phosphorescence, Fluorescence, and Chemiluminescence in Clinical Chemistry Food (Volume 5) Fluorescence Spectroscopy in Food Analysis Forensic Science (Volume 5) Fluorescence in Forensic Science Peptides and Proteins (Volume 7) Capillary Electrophoresis in Peptide and Protein Analysis, Detection Modes for Fluorescence Spectroscopy in Peptide and Protein Analysis Electronic Absorption and Luminescence (Volume 12) Absorption and Luminescence Probes Fluorescence in Organized Assemblies Fluorescence Lifetime Measurements, Applications of Indirect Detection Methods in Capillary Electrophoresis Liquid Chromatography (Volume 13) Capillary Electrophoresis

HIDC HOMO HPLC HuSCC IA ICG IEF IgG IR ISC ITP LIF LPS1 LUMO MEKC MPc MS NATO NHIgG NIR NIRCD NIRD NIRFDCD NIRFIA NIRLIF NPc Pc PCR PMMA PMO PMT PPPMO PS RIA SNR TLC USEPA UV UV/VIS VCD WHO WORM

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