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The Biomimetic Immunoassay Based on Molecularly Imprinted Polymer: A Comprehensive Review of Recent Progress and Future Prospects


  • Z.X. Xu,

    1. Authors Xu, Zhang, Chen, and Qiao are with College of Food Science and Engineering, Shandong Agricultural Univ., Taian 271018, China. Author Gao is with College of Forestry, Shandong Agricultural Univ., Taian 271018, China. Direct inquiries to author Qiao (E-mail: xgqiao@sdau.edu.cn).
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  • H.J. Gao,

    1. Authors Xu, Zhang, Chen, and Qiao are with College of Food Science and Engineering, Shandong Agricultural Univ., Taian 271018, China. Author Gao is with College of Forestry, Shandong Agricultural Univ., Taian 271018, China. Direct inquiries to author Qiao (E-mail: xgqiao@sdau.edu.cn).
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  • L.M. Zhang,

    1. Authors Xu, Zhang, Chen, and Qiao are with College of Food Science and Engineering, Shandong Agricultural Univ., Taian 271018, China. Author Gao is with College of Forestry, Shandong Agricultural Univ., Taian 271018, China. Direct inquiries to author Qiao (E-mail: xgqiao@sdau.edu.cn).
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  • X.Q. Chen,

    1. Authors Xu, Zhang, Chen, and Qiao are with College of Food Science and Engineering, Shandong Agricultural Univ., Taian 271018, China. Author Gao is with College of Forestry, Shandong Agricultural Univ., Taian 271018, China. Direct inquiries to author Qiao (E-mail: xgqiao@sdau.edu.cn).
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  • X.G. Qiao

    1. Authors Xu, Zhang, Chen, and Qiao are with College of Food Science and Engineering, Shandong Agricultural Univ., Taian 271018, China. Author Gao is with College of Forestry, Shandong Agricultural Univ., Taian 271018, China. Direct inquiries to author Qiao (E-mail: xgqiao@sdau.edu.cn).
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Abstract:  Immunoassay, based on a selective affinity of the biological antibody for its antigen, is one of the most usual analytical methods in food safety and environmental chemistry. However, it presents several drawbacks because of the nature of the antibody. Molecular imprinting technique, due to its high selectivity and stability, ease of preparation and low cost, has shown great potential in producing artificial antibodies in biomimetic immunoassays. This article focuses on the recent states, advantages, current problems and outlooks of molecularly imprinted radio, fluoro, enzyme-linked and chemiluminescent immunoassays, and biomimetic immunosensor, with special emphasis on the challenges in developing biomimetic enzyme-linked immunosorbent assays (BELISAs). The biomimetic immunoassay method will provide an important new analysis platform in food safety, although the sensitivity and specificity is relatively low.

Practical Application:  As a new simple analysis method, the biomimetic immunoassay has attractive prospect, although some limitations were existed in real-sample assay. In this critical review, some promising solutions for overcoming its drawbacks were put forward, which may promote the more quick development and extensive application of this method in food safety.


With the economic and social development, many food safety incidents had been emerged in the world. For example, U.K. Food Standards Agency announced the recall of food contaminated with the carcinogenic Sudan I in 2005. In China and some other Asian countries, it is said that the redder the egg yolk is, the more nutrient it is, Sudan IV was found in some of the red-yolk duck eggs in China in 2006 (Zhang and others 2006). Especially, the melamine incident taken place in 2008 made a severe knock on Chinese milk industry and caused panic among customers in China. For the assurance of human health, control of poison substances in adulterated foods is very crucial, and the accurate and simple analytical methods are required.

Many efforts have been devoted to detect the contaminated compounds in food samples. The analytical protocols based on gas chromatography, liquid chromatography (LC), LC coupled to mass spectrometry (LC/MS), electrochemiluminescence, amperometric acetylcholinesterase biosensor, and immunoassay methods have been developed. Among them, immunoassay is one of the most common methods (Chan 1987; Hage 1995; Fieitag 1999; Haupt 2003; Wang and others 2007) and has been widely used in clinical, agricultural, food analysis, or routine research (Milstein 1980; Lidell 2001). However, immunoassay method still has several drawbacks due to the nature of the biological antibody (Lavignac and others 2004), which are shown in Table 1. Design and synthesis of functionalized materials with antibody-like characteristic to develop a new assay format has become a longstanding goal.

Table 1–.  Advantages and limitations of MIPs compared to biological antibodies in immunoassay.
MIPsBiological antibodies
Easy for imprinting small moleculesDifficulty to produce and select antibodies for small molecules
Can be used in both aqueous and organic mediaRestricted to aqueous conditions
Synthetic: not need animalsBiological production need animals
High chemical, physical, and thermal stabilityVery fragile and unstable
Simple storage requirementsNeed to be lyophilized and may denature upon long-term storage
Preparation: fast and easyPreparation: time-consuming and difficult, monoclonal strategy allows large long-term production
Require a relatively large amount of templateRequire a relatively small amount of antigen
Controllable batch to batch reproducibilityPolyclonal antibodies are specific to each animal. Monoclonal antibodies allow batch reproducibility
Reusable for more than 100 timesNonreusable
Cost: lowCost: high
ELISA: simplified procedureELISA: time-consuming
Low sensitivityHigh sensitivity
Low specificity in aqueousHigh specificity in aqueous
High cross-reactivityLow cross-reactivity

Molecular Imprinting Technology

Among the approaches applied, one of the most interesting and promising methods is using molecular imprinting technology to prepare biomimetic mimics that can imitate the molecular recognition ability of biological antibodies (Wang and others 2007). In this process, the template is initially allowed to establish bond formation with polymerizable functionality, and the resulting complexes are then copolymerized in the presence of a large excess of cross-linkers (Allender and others 1999; Haupt and Mosbach 2000; Stephenson and Shimizu 2007), typically over 60% (Wulff 1995). Subsequent removal of the template molecule results in cavities whose shape, size, and spatial arrangement are complementary to the template. The resulting molecularly imprinted polymers (MIPs) possess high selectivity and specificity for template molecules that are similar to natural antibodies (Sellergren 1997; Haupt and Mosbach 1998).

MIPs have been reported to be utilized in many fields, such as chiral separation and chemical sensors (Wulff and others 1977; Vivek and Hunagxian 2004), and their use as sorbent material for solid-phase extraction (SPE) is potential of the most usual applications of MIP (Andersson 2000; Ramström and others 2001; Molinelli and others 2002; Francesco and others 2005; Alexander and others 2006; Christina and Hans 2006). On-line molecularly imprinted SPE (MISPE) coupled with LC had been reported, which not only have the advantages of MISPE and LC, but also can simplify the process (Wang and others 2007). However, one of the most exciting applications of this imprinted functionalized material is using as synthetic antibody in immunoassay (Ye and Haupt 2004).

Development of MIP-Based Biomimetic Immunoassays

Radio-molecularly imprinted immunoassay

The MIP-based immunoassay based on a competitive radioligand-binding format was firstly developed by Mosbach and others in 1993 (Vlatakis and others 1993), which were analogous to solid-phase sorbent assay except that the immobilized antibody was replaced with a bronchodilator theophylline or diazepam MIP. It was indicated that both polymers had comparable property to the biological antibody and exhibited the potential to substitute for antibodies in immunoassays. This method showed good correlation with that of an enzyme-multiplied immunoassay when it was used for determination of theophylline in human plasma samples.

Subsequent work for the antimorphine and antienkephalin polymers was reported (Andersson and others 1995), and the results were comparable with those obtained in biological antibody systems, which confirmed the feasibility of MIPs as antibody mimics to be used in immunoassays. Another MIP sorbent assay was reported in the case of a plethora polymer (Andersson 1996), which demonstrated greater selectivity with 1% cross-reactivity toward (R)-propranolol than that of the antibody assay with 5% to 7% cross-reactivity. This format has also been used to develop assay systems for other compounds such as drugs, herbicides, and corticosteroids (Ramström and others 1996).

Most of the labeled analytes used in biomimetic immunoassays are radioactive derivatives because of their more sensitivity. Unfortunately, radioassays, involving handling of radioactive materials and producing radioactive waste, are undesirable and their applications are limited. Therefore, development of alternative assays formats based on other labels and detection methods is increasing, such as immunoassay with enzyme reaction or fluorescence format (Hemmila 1985).

Fluoro-molecularly imprinted immunoassay

Fluorescently labeled competitors have been used to monitor binding in MIP-based immunoassay. Piletsky and others (1977) firstly described a competitive molecularly imprinted assay, in which the 5-[(4,6-dichlorotriazin-2-yl)amino]-fluorescein (DTAF), a fluorescent triazine derivative, was competed with triazine for the available binding sites, and fluorescence was shown to be proportional to triazine concentration. A immunoassay for detection of 2,4-dichlorophenoxyacetic acid (2,4-D) had been proposed by the Haupt research group based on an MIP competition with a fluorescent probe (Haupt and others 1998; Haupt 1999), which had structural similarities with analyte. The specificity and selectivity of the assay were similar to the competitive radioligand-binding assay using the same polymer and radiolabeled analyte. Recently, an automated molecularly imprinted sorbent assay for the analysis of penicillin-type β-lactam antibiotics (BLAs) has been proposed (Urraca and others 2007). Highly fluorescent competitors (emission quantum yields of 0.4 to 0.95), molecularly engineered to contain pyrene labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers, have been tested as analyte analogues in the competitive assay. The IC50 value (calculated as the concentration giving 50% inhibition of color development) of penicillin G was 1.81 × 10−6 M, and the detection limit was 1.97 × 10−7 M. Competitive binding studies demonstrated various degrees of cross-reactivity with penicillin-type BLAs from 13% to 71%. Results indicated that the total analysis time of this method was 14 min and the MIP reactor could be reused for more than 150 cycles. The accuracy of this method was tested for analysis of penicillin G in spiked urine samples with a good recovery of 92%.

Fluoro-molecularly imprinted assay may have better competition and more sensitivity, as the structure of fluorescently labeled antigent was less changed. However, the fluorescein is unstable and easy to be decompounded especially with the sunlight, and it has high fluorescent backgrounds, which may affect the detection limit and sensitivity. This method is only studied in the laboratory.

Molecularly imprinted enzyme-linked immunosorbent assay

Surugiu and others (2000) are first to develop an MIP-based immunoassay using an enzyme-labeled conjugate as competitive probe. In this research, a 2,4-D MIP microsphere was prepared by precipitation polymerization, the tobacco peroxidase (TOP) was used as the label, and the assay was developed based on either colorimetric or chemiluminescent detection. By measuring the activity of the enzyme in the supernatant, it was shown that 2,4-D was able to compete with 2,4-D-TOP for the binding sites of the MIP. Quantification of the analyte was possible at concentrations ranging from 40 to 60 μg/mL and 1 to 200 μg/mL using either colorimetric or chemiluminescence detection, respectively. The potential of the microcystin-LR MIP-based immunoassay compared to enzyme-linked immunosorbent assay (ELISA), which based on monoclonal or polyclonal antibodies, was investigated by Chianella and others (2002). The affinity of the MIP was found to be comparable to the affinity of polyclonal antibodies but lower than the one of monoclonal antibodies (Table 2).

Table 2–.  Merits and limitations of MIP-based immunoassay method in application.
MIP-based immunoassay formatMeritsLimitations
Radio-molecularly imprinted immunoassayHigh sensitivityProduce radioactive waste
Fluoro-molecularly imprinted immunoassayBetter competition and more sensitiveUnstable and easy decompounded
Molecularly imprinted enzyme-linked immunosorbent assayWide application and rapidPoor competition and high cross-reactivity
MIP-based chemiluminescence immunoassayLow detection limit, wide linear rangesPoor stability and reproduction
MIP-based biosensorSimple and rapidLow selectivity and sensitivity

As a possible solution for the challenge in biomimetic immunosorbent assay, the preparation of a functionalized membrane with high hydrophilic, accessibility, and binding ability has become interesting and attractive (Piletsky and others 2000a), and it is often necessary to control the thickness of the film in order to optimize the sensitivity of the MIP-based ELISA (Vandevelde and others 2007). Several approaches relied on awkward adaptation of bulk-polymerization techniques had been used to generate MIP films, resulting in poor control of film thickness and uniformity (Jakusch and others 1999). A thin polymer layer against epinephrine coated on microplate wells by chemical oxidative polymerization of 3-aminophenylboronic acid as a functional monomer in the catalysis of ammonium persulfate was reported (Piletsky and others 2000b; Piletsky and others 2001). An ELISA based on the free ligand and horseradish peroxidase (HRP) and norepinephrine (HRP-N) conjugate competition for MIP was presented. The high stability of the MIP and good reproducibility of the method make it present an attractive alternative to conventional ELISA for food analysis. We have recently reported a sensitive direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method based on a novel molecularly imprinted film of controlled thickness (Wang and others 2009). The prepared homogeneous film was directly synthesized on the surface of wells in 96-well plates by a room temperature ionic liquid (RTIL) mediated, chemical oxidative polymerization method in conjunction with molecular imprinting technology. Thus, additional immobilization steps are not required and plates can be used directly in ELISA applications. This BELISA method had a higher selectivity for estrone, and competitive binding studies demonstrated various degrees of cross-reactivity with five estrogenic compounds ranging from 29.9 to 46.5%. Eighty minutes of analysis time was reduced when compared to traditional ELISA, and the novel film was able to be reused more than 50 times. The IC50 and the detection limit values under optimized experimental conditions were 7.47 × 10−7 M and 2.99 × 10−8 M, respectively. This developed method was applied to the determination of estrone in spiked lake water samples and demonstrated excellent recoveries ranging from 82.0 to 95.0%.

In general, MIP can be substituted for the natural antibody used in BELISA format but remains lower binding affinity and selectivity, and the drawbacks will be progressively diminished with the development of new molecular imprinting and ELISA technology.

MIP-based chemiluminescence immunoassay

Chemiluminescence with low detection limit and wide linear ranges is attractive, and some sensitive detection methods, based on chemiluminescence immunoassay combined with the MIP, have subsequently been reported.

The chemiluminescence imaging MIP-based immunoassay analogous to competitive ELISA was firstly presented by Surugiu group (Surugiu and others 2001). In this assay, the microtiter plates were coated with 2,4-D polymer microspheres, which were fixed in place by using poly(vinyl alcohol), and the antigen of 2,4-D was labeled with the TOP. The analyte peroxidase conjugate was incubated with the free analyte in the microtiter plate, and the bound fraction of the conjugate was quantified. The chemiluminescence reaction of luminol was used for detection, and calibration curve corresponding to analyte concentrations ranging from 0.01 to 100 g/mL was obtained. Another MIP-based chemiluminescence imaging method for fast detection of dipyridamoleinreal in sample was established following the same mode with a detection limit of 0.006 μg/mL (Wang and Zhang 2008).

An MIP-based flow injection capillary chemiluminescent ELISA was developed by Surugiu and others (2001). In this mode, a glass capillary was modified by covalently attaching an imprinted polymer to the inner capillary wall, the analyte of herbicide 2,4-D was labeled with TOP, and chemiluminescence was detected using a photomultiplier tube or a CCD camera. The analyte-peroxidase conjugate was passed together with the free analyte through the polymer-coated capillary mounted in a flow system. After washing, the chemiluminescent substrate was injected and the bound fraction of the conjugate was quantified by measuring the intensity of the emitted light. Calibration curve corresponding to analyte concentrations ranging from 0.5 to 50 μg/mL was obtained.

MIP-based chemiluminescence assay can be more sensitive but the stability and reproduction is poor, which may limit the application and development of this format.

MIP-based immunosensor

One of important applications of MIP as artificial recognition element is applied in sensors, and these methods had been used widely in biological, environmental monitoring, and food process (Yano and Karube 1999; Hayden and others 2003). In which 2 formats of MIP were used, one is that the prepared MIP particle or membrane connected with the transducer, and the other format is that the imprinted membrane was directly polymerized in the transducer surface

MIP-based biosensors were rapid, convenient, and inexpensive when compared with the traditional methods, and more suitable for the on-spot detection in food safety. However, many factors will affect the measurement and further endeavor should be done to eliminate the measurement error, and the construction of a steady biosensor still remains challenge (Feng and others 2004).

Application in food safety

The developed biomimetic immunoassay is an ongoing process and few applications in food safety had been reported (Table 3). For example, a quartz crystal microbalance was employed as a sensitive apparatus of biosensor for the determination of sorbitol based on a molecularly imprinted electrosynthesized polymer (Feng and others 2004). The linear relation was 1 to 15 mM, and the detection limit was about 1 mM. Another piezoelectric sensor for caffeine detection in surface waters (Ebarvia and Sevilla 2005) or daminozide in apples (Yan and others 2007) based on MIPs had been developed, the schematic representation was depicted in Figure 1, and the detection limits were 5.9 × 10−11 mg/mL and 5.0 × 10−8 mg/mL, respectively. A novel flow chemiluminescence clenbuterol sensor based on MIP had been reported (Zhou and others 2004) and this method could be used for determination of clenbuterol in meat sample.

Table 3–.  The application of MIP-based immunoassay in food analysis.
AnalytesImmnoassay formatDetection limitRef.
SorbitolQuartz crystal microbalance sensor1 mMFeng and others (2004)
Caffeine in surface watersPiezoelectric quartz crystal sensor5.9 × 10−11 mg/mLEbarvia and Sevilla (2005)
Daminozide in applesPiezoelectric crystal sensor5.0 × 10−8 mg/mLYan and others (2007)
Clenbuterol in the urineFlow chemiluminescence sensor3.0 × 10−10 mg/mLZhou and others (2004)
EstroneBELISA2.99 × 10−8 M    Wang and others (2009)
Figure 1–.

Schematic representation of the instrumentation system of the caffeine sensor (Ebarvia and Sevilla 2005).

Food as complex matrice is hard to detect by the traditional analytical method and a complicated pretreatment procedure is needed. MIP-based biomimetic immunoassay with good accuracy and simple pretreatment procedure for determination of 2,4-D, β-lactam and penicillin G might be used to monitor the pesticides in agricultural products or antibiotics in milk and animal feed samples in the future.

Advantages of MIPs over biological antibodies in immunoassay for food detection

Compared to biological antibodies, MIPs using as biomimetic antibodies have the following advantages in immunoassays (Table 1). Firstly, the production of antibodies is laborious, and the purification is tedious, expensive, and often requires special handling methods (Brooker and others 1979). Whereas MIPs can be easily modified to increase the affinity of receptor sites, and the need to use laboratory animals for antibody production is avoided (Fischer and others 1991; Mulchandani and others 1998). Secondly, it is difficult to produce and select antibodies for small molecules. However, such compounds are best suited to molecular imprinting. Thirdly, natural antibodies are relatively unstable and very sensitive to the pH and temperature, which limits their applications under controlled conditions such as acid or alkaline foods, and their storages are difficult. MIPs are synthetic receptor sites with high physical and chemical resistance toward various external degrading factors (Matthew and other 2004) and they can be stored at ambient temperatures and in a dry state without loss of performance (Vivek and Hunagxian 2004). More importantly, MIP can be reused after extraction, and an MIP reactor can be reused for more than 100 to 150 times without significant loss (Urraca and others 2007; Wang and others 2009), thus cost per analysis of the MIP-based immunoassay is drastically reduced. Without the antibody coating and BSA/PBS blocking steps, the analysis time of the BELISA can be reduced to 80 min. The operation procedure is also simplified, which was suitable for rapid detection of food samples.

Current limitations of MIP-based immunoassays

Above studies have confirmed the feasibility of the MIP to be substituted for antibodies in immunoassays, however, there are many problems involving low sensitivity and specificity (Table 1), it is seemed to be less practical in sample analysis, and the real challenge having always been considered is to use enzyme labels (Palomo and others 1997; Haupt and others 1998). Firstly, enzymes often only work in aqueous buffers, whereas the major preparing methodology of MIP is organic-polymer-based system, the synthesis and use of MIP is restricted to organic solvents. Secondly, the rather hydrophobic nature and nonspecific binding of MIPs in the aqueous environment can affect competitive reactions, and the cross-reactivities for structural analogues were higher. Thirdly, the binding affinities and selectivity of MIPs remain lower than antibodies. It is known that most of the MIPs are polymer particles or microspheres, the highly cross-linked structure and lower affinity of the MIPs limit the access of the biological molecule to the imprinted binding sites, and slow leaching of the template molecule from the polymer matrix, which can limit the sensitivity of MIP-based immunoassay (Ellwanger and others 2001). An example is that the detection limit for the analysis of estrone by BELISA was only 100 times higher than that obtained using polyclonal antibodies as the recognition element in ELISA (Wang and others 2009). Fourthly, in competitive assay format, enzyme is a macromolecule, the structure of enzyme labeled antigen is quite different from the template molecule, which has formed a “small ball connecting with a big ball” mode, making the enzyme conjugate difficult to enter the three-dimensional cavity to combine with recognition sites of MIPs. Fifthly, lack of an inherent signaling mechanism in MIPs and poor optical and material properties of MIPs have become the major challenges in developing biomimetic biosensor format (Stephenson and Shimizu 2007). Furthermore, MIPs are usually removed after incubation by centrifugation and then the supernatant is used for colorimetric measurements, which is difficult to eliminate the interference of nonconjugated enzyme and the template, and in addition, it is hard to completely remove the template from MIPs. Finally, how to control the coordination of chemical and biochemical reaction systems and improve the stability and reproducibility is also one of the key problems for the MIP-based ELISA.

Despite the similar concept and mechanism between MIPs and biological antibodies, they exhibit different recognition properties (Umpleby and others 2000; Umpleby and others 2001). The most significant difference is that monoclonal antibodies have a single type of homogeneous distributions binding site, whereas MIPs contain a heterogeneous distribution of binding sites, which is asymmetric and asymptotic, making the MIPs have lower binding affinity and selectivity, and are only limited to certain formats (Umpleby and others 2004; Toth and others 2006). For example, the concentration-dependant binding properties can be exploited at low analyte concentrations, under these conditions, the small fraction of high-affinity binding sites become dominant and MIPs display considerably higher selectivity (Zimmerman and others 2004), which is particularly problematic in biomimetic applications. This can explain why MIPs do not show efficiencies comparable to antibodies in these applications and help to find the solutions to improve the sensitivity of biomimetic immunoassay (Szabelski and others 2002).


The preparation of MIPs compatible with aqueous environment

It is known that in aqueous solution, the specific interactions between recognition sites of MIPs and analytes are weakened, while the nonspecific interactions are strengthened, making the good recognition of MIP in aqueous environment a challenging subject (Ansell 2004). These disadvantages have greatly hindered the advancement of MIPs in BELISA and biomimetic immunosensor formats. In order to improve the sensitivity and selectivity of MIP-based immunoassay, many endeavors have been devoted to prepare an MIP capable of recognition and adsorbing template in aqueous environment (Mizutani and others 1999; Zhong and others 2001; Ersöz and others 2004; Yoo and others 2004; Yu and others 2008).

Choice of solvent

MIPs usually demonstrated their best performance in the same solvent as traditionally used for the polymer fabrication. In order to eliminate the nonspecific interactions and optimize the performance of assay in an aqueous environment, a small amount of a nonionic surfactant such as Triton X-100 or a miscible organic of ethanol was selected to lessen the surface tension and minimize hydrophobic interactions (Ansell 2004). It is pleased that the novel MIP that polymerized in aqueous solution had been reported (Mizutani and others 1999; Zhong and others 2001; Ersöz and others 2004; Yoo and others 2004; Yu and others 2008), making the MIP potential as artificial antibody in biomimetic immunoassay.

Choice of functional monomer and cross-linker

The affinity and selectivity of MIP depends on not only the size and shape of the imprinted cavity, but also the preparation and rebinding interactions, and the types of template and functional monomer will play important roles on the MIP ability (Hwang and Lee 2002). Piletsky and others (2008) investigated the ionization properties of monomeric and polymeric functional groups, and it was found that the acidic monomers were easier to form the ionic interactions with the templates than basic monomers. Ionic interactions are in theory stronger than hydrogen bonds and more resistant to the interfering impact of water molecules in molecular recognition processes. In order to maximize the role of ionic interactions, the MIP should be prepared using a strong acidic monomers such as acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) or 2-(trifluoromethy)acrylic acid (TFMAA). Metalloporphyrins and their analogues, known to recognize both hydrophilic and hydrophobic guests with significant selectivity and particular affinity in water, may offer potential resolution of the existed problems if used MAA, 4-VP or metalloporphyrins as co-monomers in the preparation of MIPs (Mizutani and others 1999).

Recently, a new approach taking form recognition sites containing both hydrophobic interactions and hydrogen bonding had been reported (Ersöz and others 2004). Methacrylamidoantipyrine that has π electron-rich aromatic ring was chosen as monomer, 4-nitrophenol which has electron-poor aromatic ring was chosen as a template molecule, and a novel MIP had been synthesized. Results indicated that the selectivity was mainly based on the affinity of shape match to the cavity created by template printing. A novel water-soluble MIP had been reported by Zhong and others (2001), in which the cross-linking agent N,Ń-diacryloylpiperazine (DAPA) was replaced with a hydrophilic monomer of ethyl 2-hydroxymethylacrylate (EHMA), and results showed that the polymer exhibited good water recognition and binding of cholesterol.

Choice of mediated template

Cyclodextrins (CDs), as one of supramolecular host compounds with a hydrophilic exterior and a hydrophobic cavity, is capable of binding the hydrophobic structures of guest (Yoo and others 2004). Using it as mediated template in molecular imprinting can help improve the recognition ability of MIP in aqueous solution. A polymerizable β-CD used as a hydrophobic moiety capable of screening electrostatic interactions between monomers and template in an aqueous environment had been reported by Piletsky and others (2008).

RTILs are interesting solvents with unique characteristics, the low vapor pressure of RTILs could assist in reducing the problem of MIP bed shrinkage and can also act as pore template in polymerization reaction (Liu and others 2005; Wang and others 2006). RTILs have been shown to accelerate the synthesis process, and improve the selectivity and adsorption of trans-asconitic acid imprinted organic polymers (Surugiu and others 2001; Schmidt and others 2004; Booker 2006). In our study, a hydrophobic RTILs of BMIM+PF6 was used to reduce the cracking and shrinking of the novel imprinted film and to improve the selectivity using in the polymerization process.

Hapten selection and synthesis

One of the key steps in development of immunoassay is designing and synthesis of hapten. The optimum hapten for a target analyte molecule should be a near-perfect mimic of that molecule in structure and geometry, in electronic and hydrogen-bonding capabilities, and in its hydrophobic properties. The spatial arrangement, nature, and size of the enzyme conjugate must be tailored to the template structure to achieve efficient competition for the MIP-binding sites. For the labeled conjugate, the one showing best performance in an MIP-based assay was the one providing the highest sensitivity in an immunoassay based on the same type of measurements (Wang and others 2009).

A significant difference between HRP-labeled conjugate and HRP binding to the novel imprinted film clearly indicated that the hapten plays an essential role on the specific recognition of MIP in BELISA (Wang and others 2009). Because the binding affinity and recognition ability of the template molecule by the MIP relied on not only the imprinted cavities, but also the template-specific binding sites, and the functional group of template has an important effect on the specificity of the assay, as it reacted with the monomer during the polymerization process.

Optimizing of the labeled conjugate

In ELISA, HRP conjugate plays an important role on the sensitivity. The amount of the HRP-labeled hapten added to each immunoassay should be the minimum so that it can be reliably quantified, and the competitive reactions will work best when 30% to 80% of the HRP-labeled template was bond to the MIPs in the absence of any analyte (Ansell 2004). The reason is that under the ideal condition, there is a shortfall of strong binding sites, which are most interested in the BELISA—if analyte or an interferent displaces the HRP-labeled hapten from these sites, it will not bind to the weaker sites, but will be truly displaced. This study is of great significance for choice of potential solution to improve the sensitivity and overcome the existed problems of BELISA.


MIPs with attractive merits can be alternated to biological antibody. However, MIP-based immunoassays developed slowly and few studies have been reported in recent years because of the significant problems and challenges, especially in enzyme-labeled formats, and it can be used in sensing applications that do not require high sensitivity and selectivity levels, especial for small molecule analysis. This new analytical methodology is at the stage of demonstrating proof-of-principle, and a number of significant issues and opportunities remain. With the molecular imprinting and immunoassay technology developed, the sensitivity and accuracy of biomimetic immunoassays improved, it will play an important role in food safety.


We are grateful for financial supports from the Natl. Natural Science Foundation of China (project nr 31071543), Chinese Postdoctoral Science Foundation Project (project nr 20090451342).