Molecularly Imprinted Materials
5.5 Selective Adsorption and Detection of Proteins
As mentioned above, the MIP‐based sensor technologies have been rapidly developed, especially in bioanalytical fields. In the near future, bio‐related NPs (such as exosome) and living cells will be able to be detected by MIP‐sensors.
Molecularly Imprinted Materials 171
core–shell MIP was endowed with a homogeneous population of high‐affinity binding sites, all having the same orientation. The MIP has no or little cross‐reactivity with other serine proteases and unrelated proteins. Interestingly, King et al. reported that a hydrogel‐based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian, and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating the selectivity [55] (Figure 5.7). Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA transla- tional regulatory event, requiring 1000‐fold less cells than current methodologies.
These results provided first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation.
Based on these achievements for the selective adsorption and detection of proteins, our interest is focused on the recognition of sugars in glycoproteins by using MIPs. The selective separation and detection of certain glycoproteins are usually required. In most cases of the detection of glycoproteins, labeling procedures with fluorescent dyes, iso- topes, etc. are carried out to detect at high selectivity and sensitivity. However, the aggregation and denaturalization of glycoproteins due to the labeling process become drawbacks for the detection in the natural form. To overcome these drawbacks, various label‐free methods have been developed and provided sensitive detection of glycopro- teins [56–61]. On the other hand, further simple and effective label‐free detection of glycoproteins without any complicated instruments is more attractive. To achieve the simple and effective detection of glycoproteins, MIPs have been widely studied. MIPs are usually employed for the separation and detection of low‐molecular‐weight com- pounds because of the rigid structure due to highly crosslinked polymers. Recently, Liu et al. achieved selective adsorptions and detections for glycoproteins via a specific inter- action between a boronic acid and a diol structure of a sugar chain in glycoproteins [62–65]. These achievements are summarized in the literature [66]. Following their results, a number of papers regarding the selective separation of glycoproteins have been also been published. These recent studies showed the possibility for the practical use of MIPs regarding the selective detection of biomolecules. Kubo et al. achieved effectively the selective adsorption of carbohydrates and glycoproteins by molecularly imprinted hydrogels (MIHs) with a poly(ethylene glycol) (PEG)‐based crosslinker and 4‐vinylphenylboronic acid [67]. In addition, an MIH with a novel boronic acid mono- mer provided selective adsorption and enabled the visible detection of fructose. Tu et al. presented an antibody‐free and enzyme‐free approach, called MIP‐based plas- monic immunosandwich assay (PISA), for fast and ultrasensitive detection of trace glycoproteins in complex samples [68]. A gold‐based boronate affinity MIP array was used to extract specifically the target glycoprotein from complex samples. After wash- ing away unwanted species, the captured glycoprotein was labeled with boronate affin- ity silver‐based Raman nanotags (Figure 5.8). Erythropoietin (EPO), a glycoprotein hormone that controls erythropoiesis or red blood cell production, was employed as a test glycoprotein in this study. Specific detection of EPO in a solution down to 2.9 × 10−14 M was achieved. Wang et al. reported pattern recognition of cells via multi- plexed imaging with monosaccharide‐imprinted QDs [69]. Imprinted with sialic acid, fucose, and mannose as templates, respectively, the QDs exhibited good specificity toward the template monosaccharides. Pattern recognition constructed using the
Purification of ribosome template
Ribosome 40S
60S mRNA
Polymerize acrylamide gel to form cast around template
Break cast and remove template
Rebind template from cell extract
Identify RNAs RT-qPCR
5 4 3 2 1
Figure 5.7 Schematic overview of R‐MIP preparation. First, ribosomes are isolated from HeLa cells cytoplasmic extract using a sucrose cushion. Second, the ribosome template is combined with a mixture of acrylamide (AA) and N,N′‐methylenebisacrylamide (MBAm) monomers, and polymerization is induced under gaseous nitrogen upon addition of the initiator ammonium persulfate (APS) and the catalyst N,N,N′,N′‐tetramethylethylenediamine (TEMED). Third, the hydrogel is granulated by passing through a sieve mesh, and the ribosome template is removed from the MIP. This results in a slurry of heterogeneous PAA fragments, with cavities possessing the potential to recognize more template, based both upon three‐dimensional structure and direct interactions between the template and chemical groups on the surfaces of the cavities. Fourth, MIPs are combined with cellular extracts to capture ribosomes and associated mRNAs. Fifth, ribosome associated mRNAs are isolated from the MIP for further analysis, such as reverse transcription (RT)‐quantitative PCR (qPCR). Source: Reprinted from Reference [55], © Nature Group. Reproduced with permission of Macmillan Publishers.
Molecularly Imprinted Materials 173
intensities of multiplexed imaging unveiled the similarities and differences of different cell lines, allowing for the recognition of not only cancer cells from normal cells but also cancer cells of different cell lines. Muhammad et al. reported a new type of molecularly imprinted plasmonic substrate for a rapid and ultrasensitive plasmonic immunosand- wich assay of trace glycoproteins in complex real samples [70]. The substrates were fabricated from glass slides, first coated with a self‐assembled monolayer (SAM) of gold NPs and then molecularly imprinted with organo‐siloxane polymer in the presence of template glycoproteins. Alkaline phosphatase (ALP) and α‐fetoprotein (AFP), glycopro- teins that are routinely used as disease markers in clinical diagnosis, were used as representative targets. The LOD was 3.1 × 10−12 M for ALP and 1.5 × 10−14 M for AFP, which is the best among the PISA approaches reported.
As shown above, a variety of MIPs have been developed for the selective adsorption and detection of proteins/glycoproteins. The selectivity of these materials has been close to that of the real antibodies and we expect these intelligent materials will be uti- lized practically for living cell detections and clinical diagnosis.