Documente Academic
Documente Profesional
Documente Cultură
pubs.acs.org/jchemeduc
INTRODUCTION
THEORY
When incident light propagates in a medium of relatively higher
refractive index to a medium of lower refractive index, the ray of
light tends to reect as opposed to refract. In refraction, the
light ray changes direction and bends as it passes through two
media. In reection, the light beam rebounds after impinging
on a surface with angles of incident and reection being the
same. When the light beam seizes to cross the boundary and
is entirely reected, a total internal reection (TIR) occurs.
A common example of TIR is in professionally cut diamonds
where it renders their maximum sparkle. TIR is also critical in
the operation of ber optics where light travels along the optical
ber, reecting o its walls, within the core of the cable with
minimal loss.
During the occurrence of TIR at the interface between the
two nonabsorbing media, the fully reected light beam leaks
some electrical eld intensity into the medium that has the
lower refractive index. The leaked electrical eld is referred to
as the evanescent eld. The evanescent eld waves amplitude
decays with distance from the interface in an exponential fashion. In
SPR, the evanescent wave excites electrons within the metal layer of
a metaldielectric (the two nonabsorbing media with dierent
refractive indices) interface, yielding surface plasmons. Surface
plasmons or surface plasmon polaritrons are electromagnetic
Article
INSTRUMENTATION
Generally, there are three components to an SPR instrument
(Figure 1). One critical component is the optical light source
Article
Figure 3. Representative sensorgrams obtained from actual experiments involving two dierent murine monoclonal antibodies (mAb),
denoted as mAb-1 and mAb-2, immobilized on a carboxymethylated
dextran. The proprietary protein antigen (Ag) was passed over each
immobilized mAb chip in separate runs. In each experiment, dierent
dilutions (typically at least 45 concentrations, plus a baseline run) of
the Ag were used to determine KD values (3240 nM). A simple 1:1
interaction model (i.e., mAb + Ag mAbAg) with binding kinetics
of pseud-orst-order was applied. The time axis in a sensorgram is
expressed in seconds.
in SPR experiments include association rate constant (ka) or onrate (kon) in M1 s1, dissociation rate constant (kd) or o-rate
(koff) in s1, equilibrium association constant (KA; KA = ka/kd)
205
Article
Biacore 3000
ProteOn XPR36
Biacore T200
Sample Handling
Autosampler
Auosampler
Molecular Weight
Detection
>180 Da
>201 Da
Analytical Performance
1 pM
10 pM
20200 L
1.331.40 RIU
1.331.37 RIU
1.331.40 RIU
Analysis Temperature
1540 C
In-Line Reference
Subtraction
Yes
Yes
Automatic
Dimension (L W H)
Electric Voltage
100120 V; 220240 V
100240 V
Power Consumption
Max 580 VA
800 W
Net Weight
50 kg/110 lbs
85 kg/187 lbs
60 kg/132 lbs
Comments
http://www.biacore.com
http://www.bio-rad.com
http://www.biacore.com
SensiQ
Reichert Technologies
Cole-Parmer
a
Instrument Modelsa
http://www.biacore.com
http://www.bio-rad.com
http://www.horiba.com
http://www.bionavis.com
http://www.biosensingusa.com
http://www.plexera.com
http://www.discoversensiq.com
http://www.reichert.com/life_sciences.cfm
http://www.coleparmer.com
Article
COMMERCIAL EQUIPMENT
A number of vendors manufacture commercial SPR instruments (Tables 1 and 2),30 with estimated costs varying between
$35,000 to $300,000, depending on the application, choice of
accessories, and model. We envision that in some instances, an
academic discount could apply; used demo-units could perhaps
be obtained at a reduced price. The utility of an SPR unit
in upper-level undergraduate as well as graduate laboratories
is immense and cannot be overstated. GE Healthcare (which
acquired Biacore in 2006) and Bio-Rad Laboratories are among
the SPR manufacturers (Table 1 and 2). For example, Figure 4
shows two examples of SPR biosensor chips that are available commercially. The biosensor chips are costly (e.g., ranging
from $55 to $500 each) yet in most cases can be regenerated
and reused. In addition, running buers and immobilization
Figure 5. A simplied representative workow for a three-part laboratory session on SPR spectroscopy.
207
Article
solutions are needed for routine use. In general, the SPR instruments are robust and seldom break down under proper care
and clean up. In larger academic institutions, the SPR spectroscopy facility is used for teaching and research and often
is self-funded by fees generated for sample analysis (e.g., $35/h
to $125/h).
A quick search of the National Science Foundation (NSF)
Award database revealed a number of seed grants ($100,000
and up) in support of acquisition of a SPR instrument. The
sources of the SPR instrument funding were programs such as
Major Research Instrumentation, as well as others.
AUTHOR INFORMATION
Corresponding Author
*E-mail: rbakhtiar@msn.com.
Notes
REFERENCES
(1) Hearty, S.; Conroy, P. J.; Ayyar, B. V.; Byrne, B.; OKennedy, R.
Expert Rev. Vaccines 2010, 9, 645664.
(2) Wolf, L. K.; Gao, Y.; Georgiadis, R. M. J. Am. Chem. Soc. 2007,
129, 1050310511.
(3) Ramakrishnan, M.; Kandimalla, K. K.; Wengenack, T. M.;
Howell, K. G.; Poduslo, J. F. Biochemistry 2009, 48, 1040510415.
(4) Hendrix, M.; Priestley, E. S.; Joyce, G. F.; Wong, C.-H. J. Am.
Chem. Soc. 1997, 119, 36413648.
(5) Salamon, Z.; Wang, Y.; Brown, M. F.; Macleod, H. A.; Tollin, G.
Biochemistry 1994, 33, 1370613711.
(6) Rao, J.; Yan, L.; Xu, B.; Whitesides, G. M. J. Am. Chem. Soc. 1999,
121, 26292630.
(7) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am.
Chem. Soc. 2003, 125, 61406148.
(8) Mason, S.; La, S.; Mytych, D.; Swanson, S. J.; Ferbas, J. Curr. Med.
Res. Opin. 2003, 19, 651659.
(9) Jung, L. S.; Shumaker-Parry, J. S.; Campbell, C. T.; Yee, S. S.;
Gelb, M. H. J. Am. Chem. Soc. 2000, 122, 41774184.
(10) Cooper, M. A. Nat. Rev. Drug Discovery 2002, 1, 515528.
(11) Homola, J. Chem. Rev. 2008, 108, 462493.
(12) Schuck, P. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 541
566.
(13) Kausaite, A.; van Dijk, M.; Castrop, J.; Ramanaviciene, A.;
Baltrus, J. P.; Acaite, J.; Ramanavicius, A. Biochem. Mol. Biol. Educ.
2007, 35, 5763.
(14) Jez, J. M.; Schachtman, D. P.; Berg, R. H.; Taylor, C. G.; Chen,
S.; Hicks, L. M.; Jaworski, J. G.; Smith, T. J.; Nielsen, E.; Pikaard, C. S.
Biochem. Mol. Biol. Educ. 2007, 35, 410415.
(15) Brauner, A.; Carey, J.; Henriksson, M.; Sunnerhagen, M.;
Ehrenborg, E. Biochem. Mol. Biol. Educ. 2007, 35, 187192.
(16) Witherow, D. S.; Carson, S. Biochem. Mol. Biol. Educ. 2011, 39,
300308.
(17) Thillaivinayagalingam, P.; Gommeaux, J.; McLoughlin, M.;
Collins, D.; Newcombe, A. R. J. Chromatogr., B 2010, 878, 149153.
(18) Willander, M.; Al-Hilli, S. Methods Mol. Biol. 2009, 544, 201
229.
(19) Pattnaik, P. Appl. Biochem. Biotechnol. 2005, 126, 7992.
(20) Kodoyianni, V. BioTechniques 2011, 50, 3240.
(21) Nguyen, B.; Tanious, F. A.; Wilson, W. D. Methods 2007, 42,
150161.
(22) Myszka, D. G. J. Mol. Recognit. 1999, 12, 279284.
(23) Kuroki, K.; Maenaka, K. Methods Mol. Biol. 2011, 748, 83106.
(24) Jason-Moller, L.; Murphy, M.; Bruno, J. Curr. Prot. Prot. Sci.
2006, Chapter 19 (Supplement45), No. Unit 19.13.119.13.14.
(25) Vikholm, I.; Viitala, T.; Albers, W. M.; Peltonen, J. Biochim.
Biophys. Acta 1999, 1421, 3952.
(26) Jung, L. S.; Shumaker-Parry, J. S.; Campbell, C. T.; Yee, S. S.;
Gelb, M. H. J. Am. Chem. Soc. 2000, 122, 41774184.
(27) Stenlund, P.; Babcock, G. J.; Sodroski, J.; Myszka, D. G. Anal.
Biochem. 2003, 316, 243250.
(28) Karlsson, R. J. Mol. Recognit. 2004, 17, 151161.
(29) Roos, H.; Karlsson, R.; Nilshans, H.; Persson, A. J. Mol. Recognit.
1998, 11, 204210.
(30) Rich, R. L.; Myszka, D. G. Anal. Biochem. 2007, 361, 16.
(31) Buijs, J.; Franklin, G. C. Briefings in Funct. Genomics Proteomics
2005, 4, 3947.
208
Article
(32) Huang, X.; Wang, S.; Shan, X.; Chang, X.; Tao, N. J. Electroanal.
Chem. 2010, 649, 3741.
(33) Du, M.; Zhou, F. Anal. Chem. 2008, 80, 42254230.
(34) Tang, Y.; Zeng, X.; Liang, J. J. Chem. Educ. 2010, 87, 742746.
209