Report
Over the past two decades, enormous activity has taken place in thefieldof sensor technology. Biosensors, in particular, have attracted considerable attention because of their extraordinary sensitivities and specificities. However, such devices often lack storage and operational stability because they are based on a fragile biological recognition element an enzyme or antibody. For this reason, biosensors have not become quite the commercial success expected in the early euphoric development phase. An emerging technology called molecular imprinting, however, could provide an alternative. This technique leads to highly stable synthetic polymers that possess selective molecular recognition properties because of recognition sites within the polymer matrix that are complementary to the analyte in the shape and positioning of functional groups. Some of these polymers have high selectivities and affinity constants comparable with naturally occurring recognition systems such as monoclonal antibodies or receptors which make them especially suitable as constituents in chemical (biomimetic) sensors for analytical chemistry. Plastics that possess a molecular memory Molecular recognition between a molecular receptor (host) and a substrate (guest) in a matrix containing structurally related molecules requires discrimination and binding; this can happen only if the binding sites of the host and guest molecules complement each other in size, shape, and chemical functionality. Biological systems, such as enzyme-substrate, antibody-antigen, and hormone-receptor systems, demonstrate molecular recognition properties that have developed by natural selection. Chemical recognition systems, however, have been developed mainly by rational design in the laboratory, although combinatorial approaches, which can generate recognition systems by selection Dario Kriz Olof Ramström Klaus Mosbach Lund University (Sweden) 0003-2700/97/0368-345A/$14.00/0 © 1997 American Chemical Society
MOLECULAR IMPRINTING
New Possibilities
for Sensor Technology Molecular imprinting-based biomimetic sensors could drovide an alternative to often unstable biosensors for industry, medicine, and environmental analysis from large libraries, will become increasingly important in the future. The principles of host-guest chemistry were set out by Cram, Lehn, and Pedersen in die 1960s and 1970s and have been developed for a multitude of synthetic or semisynthetic systems such as crown ethers, cyclodextrins, and cyclophanes (1-3). Such hostguest systems are potentially very useful as recognition elements in analytical applications and have been used in separation and isolation processes. One of the most intriguing areas for host-guest chemistry is the development of biomimetic recognition systems. A wide range of analytical procedures depend on reliable and sensitive biological recognition elements such as antibodies and enzymes. Because such biomolecules can suffer from stability problems, syn-
thetic counterparts are desirable. One such approach to biomimetic recognition is the fabrication of molecularly imprinted polymers (MIPs) (4). Molecular imprinting is a powerful method for preparing synthetic recognition sites with predetermined selectivity for various substances. Although the concept of molecular imprinting has existed for many years (5), it is only recently— accelerated in part by a report on "plastic antibodies" (6) that we have witnessed a surge in interest. Molecular imprinting can be approached in two ways: the selfassembly approach (7) and the nized approach (8) (Figure 1). These two approaches which differ with respect to the interaction mechanism in prepolymerization follow common molecular recognition terminology (1 9)
Analytical Chemistry News & Features, June 1, 1997 3 4 5 A
Report bonds that hold the print molecules to the macroporous polymer matrix, recognition sites complementary to the analyte remain in the polymer. The recognition properties of MIPs are highly attractive. In several cases, the selectivities and affinities acquired from the molecular imprinting process are on par with natural binding entities, such as antibodies. In self-assembly imprinting protocols using only noncovalent interactions, which have been predominantly used to produce biomimetic matrices, the principal means for exerting specificity are ionic interactions and hydrogen bonding between the analytes and die polymer functional groups, much akin to natural systems. In addition to these polar interactions the three-dimensional geometry of die sites contributes to the overall quality of the specificitv Until recently hydrophobic interactions of paramount importance in biological systems have been used rather haphazardly Although hydrophobic forces are potentially more difficult to master because thev are less specific and have less direction comhi nations of nolar and hydrnnhobic interar tions may, in the future, be used to gener4.
Figure 1 . Principles of molecular imprinting. (a) Self-assembly approach, (b) Preorganized approach.
The self-assembly molecular imprinting approach involves host-guest complexes produced from weak intermolecular interactions (such as ionic or hydrophobic interactions, hydrogen bonding, and metal coordinations) between the analyte and the monomer precursors. These self-assembled complexes are spontaneously established in the liquid phase and are then sterically fixed by polymerization with a high degree of crosslinking. After removal of the print molecules from the resulting macroporous matrix vacant recognition sites that are specific to the print molecule are established The shape of the sites maintained by the polymer backbone and the arrangement of the functional crrnups in the rprnpnition sites rpsults in affinity analyte Figure 2 shows an example of the polymerization chemistry used to pre346 A
pare MIPs that are selective for dansy--Lphenylalanine. A combination of carboxylateand pyridinyl-containing monomers (methacrylic acid and vinylpyridine) prearranges with the amino acid derivative in acetonitrile solution before polymerization. Following crosslinking with ethylene glycol dimethacrylate by free-radical polymerization, a rigid polymer is produced that, after extensive washing to remove the print molecule retains recognition sites specific for dansyl-L-phenylalanine. The preorganized molecular imprinting approach involves formation of strong reversible covalent arrangements (e g boronate esters imines and ketals) of the monomers with the print molecules before polymerization Thus the print molecules need to be "derivatized" with HIe
monomers before the actual imprinting is performed After cleaving the covalent
Analytical Chemistry News & Features, June 1, 1997
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ate strong and selective binding. In this way, even more improved biomimetjc matrices may be produced. So far, MIPs have been prepared with affinities for proteins, amino acid derivatives, sugars and their derivatives, vitamins, nucleotide bases, pesticides, and pharmaceuticals (e.g., theophylline, morphine, diazepam, naproxen, Cortisol, and pentamidine) (8). The binding of some of these MIPs (also referred to as antibody binding mimics) has been comparable with the binding of some natural monoclonal antibodies (6,10). One of the advantages of molecular imprinting is that imprints can be made of compounds against which it is difficult if not impossible to raise anttbodies The use of animals often necessary with antibodv production is avoided and the scale-un for bulk manufacture is easilv done Thus benefits are reaped from nractical ethical and economical noints of view Other advantages of MIPs are their long-term stability and resistance to chemically harsh environments (11). So far, MIPs have been used primarily as stationary phases in HPLC. Recently, however, they have been used in TLC (12), CE (13), heterogeneous binding assays (6,
Table 1 . Analytes and tranducers used in biomimetic sensors based on MIPs. Analyte
Range examined (pg/mL)
Transducer
Vitamin K, Phenylalanine anilide
0-4 Qualitative 33-3300 0-10 0-30 0-0.5 0-400
Ellipsometry Capacitance Potentiometry Amperometry Fiber-optic fluorescence Conductometry Conductometry
22 21
0-3
Optical fluorescence
30
Morphine Dansyl-Lphenylalanine Atrazine Benzyltriphenylphosphonium ions Sialic acid
14), and in biomimetic affinity sensors. A challenge that has yet to be met is improving the rather slow binding kinetics that have been observed, particularly in sensor applications.
Reference 16 20 17 11 23
played in a suitable form. The successful performance of a chemical sensor depends on the appropriate choice of recognition element and transducer. Sensor performance is characterized by selectivity, sensitivity, stability, and
reusability. Selectivity, a measure of how well a chemical sensor discriminates between the analyte and compounds of similar, or different, chemical structure, is principally determined by the recognition component within the sensor device. In this context, recognition elements of biological origin, such as enzymes and antibodies, are especially promising. Sensitivity is determined by the recognition element and the transducer. Depending on the S/N, additional amplification steps can enhance sensitivity and lower the detection limit of the analyte. Maintaining long-term stability, withstanding harsh chemical environments, and operating at high temperatures and/or pressures are severe challenges for sensors, particularly when the recognition element is of biological origin. This, in part, slowed down the application of biosensors.
Chemical sensors
Chemical sensors provide an analytically powerful and inexpensive alternative to conventional technologies by enabling the identification of a target molecule in the presence of numerous interfering species. Methodologies have been developed for many different target species (15), including gaseous substances such as anesthetics, respiratory by-products, inflammables, toxics, and nerve gases; metabolites (glucose, urea, hormones, steroids, drugs of abuse); ions (H\ Na"1", Ca2+, heavy metals) ; toxic organic vapors (benzene ,oluene) ; and proteins and microorganisms (viruses bacteria parasites) A chemical sensor selectively recognizes a target molecule in a complex matrix and generates an output signal using a transducer that correlates to the concentration of the analyte (Figure 3). The recognition element is responsible for the selective binding (and in some cases, conversion) of the analyte in a matrix containing both related and unrelated compounds. Upon binding, the transducer translates the chemical event into a quantifiable output signal. When the analyte interacts with the recognition element a change in physicochemical parameters associated with the interaction occurs This change may produce ions electrons gases heat mass changes or light and the transducer these parameters into an elprrrical outpiit clgnal
that can be amplified processed and dis-
Figure 2. Example of t h e polymerization chemistry used to prepare MIPs selective for dansyl-L- phenylalanine. Analytical Chemistry News & Features, June 1, 1997 3 4 7 3
Report
Figure 3. S c h e m a t i c of a c h e m i c a l sensor, c h a r a c t e r i z e d by a r e c o g n i tion element and a transducer close t o each other.
A n e w g e n e r a t i o n of b i o s e n s o r s
MIPs have unique properties that make them especially suitable for sensor technology. They exhibit good specificity for various compounds of medical, environmental, and industrial interest; and they have excellent operational stability. Their recognition properties are unaffected by acid, base, heat, or organic phase treatment (11), making them highly suitable as recognition elements in chemical sensors (Table 1). During some early attempts at using MIPs in sensors, optical (ellipsometric) measurements on thin layers of vitamin K imprinted polymers performed (16) Other biosensorlike approaches include the measurement of changes in the electric streaming potential over an HPLC column packed with a phenylalanine anilide-specific polymer as well as permeability studies of MIP membranes (17 18) However these arrangements do not fulfill the criterion that a biosensor have the recognition plpment and transducer in close proxim lty, they cannot be considered biomimetic sensors in the strict sense. We proposed a "real" biosensor based on an MIP in 1991 (19). This was followed by the first reported attempt to make a biomimetic sensor based on capacitance measurements on afield-effecttransistor coated with a phenylalanine anilide348 A
imprinted polymer (20). However, the results were qualitative. A subsequently described amperometric morphine sensor showed more quantitative results, enabling morphine detection in the concentration range of 0.1-10 ug/mL (11). It also showed long-term stability, resistance to harsh chemical environments, and the ability to be autoclaved—highly attractive features not normally associated with conventional biosensors. In another approach based on conductometric measurements (21), a direct signal is obtained on binding (due to the increased local concentration) of the positively charged species to the negatively charged imprints placed on the conductometric transducer. The difference in signal between the sensor and a reference sensor correlated well with the concentration of the analyte. This type of sensor arrangement is useful only in well-defined (pure) matrices in which the interference caused by conductivity of the solution can be controlled. Atrazine sensing by MIP membrane permeation measurements has also been reported (22) To date, the most convincing demonstration of the usefulness of a "real" biomimetic sensor based on molecular imprints is an optical-fiber-like device in which a fluorescent amino acid derivative (dansylL-phenylalanine) binds to the polymer particles, resulting in fluorescent signals that vary as a function of the concentration of the derivative (Figure 4) (23). Chiral selectivity was shown by using the corresponding D-enantiomer as a control.
binding capacity and response time. Using smaller polymer particles or thin polymer layers should improve diffusion rates and thus the apparent binding kinetics, giving far lower response times. Alternatively, the initial binding (pre-equilibrium) rate could be used to determine analyte concentration. Biosensors based on enzymes have, in some cases, been shown to be superior in sensitivity to affinity-based biosensors. This tendency is explained by analyte conversion, which occurs after the initial binding step in conjunction with turnover amplification and makes it possible to obtain highly sensitive amperometric transducers that are also less sensitive to unspecific binding interference. Biomimetic sensors containing catalytically active polymers should exhibit promising sensor characteristics, although so far only modest catalytic activity has been reported. Molecular imprinting of substances that resemble reaction transition states has led to polymers exhibitinff some esterolytic activity (24); other examples of catalytic reactions include the B-elimination of HF from4-fluoro-4-rt-nitronhenvn-2-butanone (25 26) and aldol condensation (27) Conducting polymers have been used as rudimentary selective partitioning phases on electrodes and have been shown to retain a "memory" for the anion
Future prospects
Rapid developments in electronics have led to microprocessors that are suitable for use in chemical sensors. Such microprocessors offer advanced signal-processing capability, and integrating the controller with the transducer could minimize noise and result in improved sensor performance. A problem when making measurements with MlP-based biomimetic sensors is the long response time (15-60 min). This delay could be minimized by optimizing the kinetics and selectivity of polymers. It is believed that the use of highly rigid polymers favors selectivity (because of the higher in-out energy barriers to exchange the analyte) and increasing response time. Similarly, polymer porosity increases polymer-
Analytical Chemistry News & Features, June 1, 1997
Figure 4. B i o m i m e t i c s e n s o r in w h i c h a n MIP s e l e c t i v e f o r t h e fluorescently labeled amino acid dansyl-L-phenylalanine w a s applied a s a layer o n t h e t i p of a f i b e r - o p t i c sensing device.
What's new that was used for doping. This ion-sieving effect has been studied amperometrically and potentiometrically and can be correlated to the ionic radius and the charge of the tested anion (28). Materials exhibiting predetermined molecular recognition selectivity in combination with electrical conductivity could be used in electrochemical sensors and would provide the basis for a new line of sensor development introducing new fusion materials constituting an integrated recognition element and transducer. Along these lines, the preparation and characterization of composite particles containing an electrically conducting polymer (polypyrrole) and an MIP for morphine have been reported (29). The morphinespecific molecular recognition properties were not significantly altered by the manufacturing procedure, which involved rather harsh treatment; and the composite particles were shown to be electrically conductive when examined as dry layers on an interdigitated finger array device. Such particles were immobilized by simultaneous electropolymerization of ovrrole onto gold-covered silica substrates and the topotrraphy of the substrates studied by atomic force microsconv This demonstrates how readily the mmnnsite particles can be electrically connected to an elprrrnrlp therpHv oh+ninino' intporation the transrlncer anri the rernoni The current generation of MIP biomimetic sensors is 100- to 1000-fold less sensitive than other types of biosensors. Although further improvements in MIPs are likely to decrease the sensitivity gap and lead to useful applications, biomimetic and other types of biosensors will most probably find their own niches in the future.
(7) Lindsey, J. S.NewJ. Chem. 1991,15, 153-80. (8) Mosbach, K; Ramstrom, O. Bio/Technology 1996,14,163-70. (9) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995,34,1812-32. (10) Andersson, L I.; Miiller, R.; Vlatakis, G.. Mosbach, K. Proc. Natl. Acad. Sci. USA 1995,92,4788-92. (11) Kriz, D.; Mosbach, K. Anall Chim. Acta 1995,300,71-75. (12) Kriz, D.; Berggren Kriz, C; Andersson, L; Mosbach, K Anal. Chem. 1194,66, 2636-39. (13) Nilsson, K; Iindell, J.; Noorrow, O.; Sellergren, B.J. Chromatogr. A1994,680,57-61. (14) Muldoon, M.; Stanker, L.J. Agric. Food Chem. 1995,43,1424-27. (15) Janata, J.; Josowicz, M.; DeVaney, D. M. Anal. Chem. 1199 46,207 R-28 R. (16) Andersson, L; Mandenius, C; Mosbach, K Tetrahed. Lett. 1988,29,5437-40. (17) Andersson, L I.; Miyabayashi, A; O'Shannessy, D. J.; Mosbach, K.J. Chromatogr. .990,516,323-31. (18) Piletsky, S.; Parhometz, Y.; Lavryk, N.; Panasyuk, T.; El'skaya, A Sens. Actuators B 1994,18-19, 629-31. (19) Mosbach, K; Andersson, L I. Swedish Patent Application, 1991. (20) Hedborg, E.; Winquist, F.; Lundstrbm, I.; Andersson, L.; Mosbach, K Sens. Actuators A 1993,37, 796-99. (21) Kriz, D.; Kempe, M.. Mosbach, K Senss Actuators B1996,33,178-81. (22) Piletsky, S. A; Piletskaya, E. V.; Elgersma, A V.; Yano, K; Karube, I.; Parhometz, Y P.; El'skaya, A. V. Biosens. Bioelectron. .195,10,959-64. (23) Kriz, D.; Ramstrom, O.. Svensson, A; Mosbach, KAnal. Chem. 1995,67, 2142-44. (24) Robinson, D. K; Mosbach, K.J. Chem. Soc, Chem. Commun. 1989814,969-70. (25) Miiller, R; Andersson, L I.; Mosbach, K Makromol. Chem., Rapid Commun. 1993,14,637-41. (26) Beach, J. V.. Shea, K J./. Am. Chem. Soc. 1994,116,379-80. (27) Matsui, J.; Nicholls, I. A; Mosbach7 K. / Org. Chem. 1996, 6,, 5454-17. (28) Wang, J.; Chen, S. P.; Lin, M.J. Electroanal. Chem. 1989,273,231—42. (29) Kriz, D.; Andersson, L I.; Khayyami, M.; Danielsson, B.; Larsson, P-O.; Mosbach, K Biomimetics s995,3,81-90. (30) Piletsky, S. A et al. Anall Lett. 1996,29, 157-70.
Klaus Mosbach, Professor of Pure and Applied Biochemistry, focuses on molecular imprinting, biomolecule immobilization, References affinity techniques, and sensor technology. (1) Cram, D. J. Angew. .hem.t Int. Ed. Engl. Dario Kriz, Senior Research Associate, spe1988,27,1009-20. cializes in sensor technology ynd electro(2) Lehn, J-M. Angew. .hem., Int. Ed. Engl. chemistry. Olof Ramstrom, Senior Research 1988,27, 89-112. Fellow, focuses on molecular imprinting (3) Pedersen, C.J. Angew. .hem., Int. Ed. and combinatorial chemistry. Address corEngl. 1988,27,1021-27. (4) Ansell, R. J.; Kriz, D.; Mosbach, K Curr. respondence to Mosbach ht Pure end ApOpin. Biotechnol. 1996, 6, 89-94. plied Biochemistry Centre for Chemistry (5) Pauling, L.J. Expl. Med. 1942, 76,211— and Chemical Engineering Lund Univer20. sity P.O. Box 124 S-22100Lund Sweden (6) Vlatakis, G.; Andersson, L I.; Miiller, R.; Mosbach, K. Nature 1993,361, 645547. (
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We are grateful to Richard Ansell and Peter Cormack for their linguistic advice.
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