Protein Determination with Molecularly Imprinted Polymer Recognition Combined with Birefringence Liquid Crystal Detection.

Liquid crystal-based sensors offer the advantage of high sensitivity at a low cost. However, they often lack selectivity altogether or require costly and unstable biomaterials to impart this selectivity. To incur this selectivity, we herein integrated a molecularly imprinted polymer (MIP) film recognition unit with a liquid crystal (LC) in an optical cell transducer. We tested the resulting chemosensor for protein determination. We examined two different LCs, each with a different optical birefringence. That way, we revealed the influence of that parameter on the sensitivity of the (human serum albumin)-templated (MIP-HSA) LC chemosensor. The response of this chemosensor with the (MIP-HSA)-recognizing film was linear from 2.2 to 15.2 µM HSA, with a limit of detection of 2.2 µM. These values are sufficient to use the devised chemosensor for HSA determination in biological samples. Importantly, the imprinting factor (IF) of this chemosensor was appreciable, reaching IF = 3.7. This IF value indicated the predominant binding of the HSA through specific rather than nonspecific interactions with the MIP.

Selective PQQPFPQQ Gluten Epitope Chemical Sensor with a Molecularly Imprinted Polymer Recognition Unit and an Extended-Gate Field-Effect Transistor Transduction Unit.

A molecularly imprinted polymer (MIP) recognition system was devised for selective determination of an immunogenic gluten octamer epitope, PQQPFPQQ. For that, a thin MIP film was devised, guided by density functional theory calculations, and then synthesized to become the chemosensor recognition unit. Bis(bithiophene)-based cross-linking and functional monomers were used for this synthesis. An extended-gate field-effect transistor (EG-FET) was used as the transduction unit. The EG-FET gate surface was coated with the PQQPFPQQ-templated MIP film, by electropolymerization, to result in a complete chemosensor. X-ray photoelectron spectroscopy analysis confirmed the presence of the PQQPFPQQ epitope, and its removal from the MIP film. The chemosensor selectively discriminated between the octamer analyte and another peptide of the same number of amino acids but with two of them mismatched (PQQQFPPQ). The chemosensor was validated with respect to both the PQQPFPQQ analyte and a real gluten extract from semolina flour. It was capable to determine PQQPFPQQ in the concentration range of 0.5-45 ppm with the limit of detection (LOD) = 0.11 ppm. Moreover, it was capable of determining gluten in real samples in the concentration range of 4-25 ppm with LOD = 4 ppm, which is a value sufficient for discriminating between gluten-free and non-gluten-free food products. The gluten content in semolina flour determined with the chemosensor well correlated with that determined with a commercial ELISA gluten kit. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the epitope sorption data. The sorption parameters determined from these isotherms indicated that the imprinted cavities were quite homogeneous and that the epitope analyte was chemisorbed in them.

Post-imprinting modification: electrochemical and scanning electrochemical microscopy studies of a semi-covalently surface imprinted polymer.

Herein we described a post-imprinting modification of the imprinted molecular cavities for electrochemical sensing of a target protein. Imprinted molecular cavities were generated by following the semi-covalent surface imprinting approach. These mesoporous cavities were modified with a ferrocene 'electrochemical' tracer for electrochemical transduction of the target protein recognition. Electrochemical sensors prepared after post-imprinting modification showed a linear response in the concentration range of 0.5 to 50 µM. Chemosensors fabricated based on capacitive impedimetric transduction demonstrated that imprinted molecular cavities without post-imprinting modification showed better selectivity. Scanning electrochemical microscopy (SECM) was used for the surface characterization of imprinted molecular cavities modified with ferrocene electrochemical tracers. SECM analysis performed in the feedback mode monitor changes in the surface state of the ferrocene-modified polymer film. The kinetics of the mediator regeneration was almost 1.8 times higher on the non-imprinted surface versus the post-imprinting modified molecular imprinted polymer.

Molecularly imprinted polymer-based extended-gate field-effect transistor (EG-FET) chemosensor for selective determination of matrix metalloproteinase-1 (MMP-1) protein.

A conducting molecularly imprinted polymer (MIP) film was integrated with an extended-gate field-effect transistor (EG-FET) transducer to determine epitopes of matrix metalloproteinase-1 (MMP-1) protein biomarker of idiopathic pulmonary fibrosis (IPF) selectively. Most suitable epitopes for imprinting were selected with Basic Local Alignment Search Tool software. From a pool of MMP-1 epitopes, the two, i.e., MIAHDFPGIGHK and HGYPKDIYSS, the relatively short ones, most promising for MMP-1 determination, were selected, mainly considering their advantageous outermost location in the protein molecule and stability against aggregation. MIPs templated with selected epitopes of the MMP-1 protein were successfully prepared by potentiodynamic electropolymerization and simultaneously deposited as thin films on electrodes. The chemosensors, constructed of MIP films integrated with EG-FET, proved useful in determining these epitopes even in a medium as complex as a control serum. The limit of detection for the MIAHDFPGIGHK and HGYPKDIYSS epitope was ∼60 and 20 nM, respectively. Moreover, the chemosensors selectively recognized whole MMP-1 protein in the 50-500 nM concentration range in buffered control serum samples.

Peptide Selection of MMP-1 for Electrochemical Sensing with Epitope-Imprinted Poly(TPARA-co-EDOT)s.

Instead of molecularly imprinting a whole protein molecule, imprinting protein epitopes is gaining popularity due to cost and solubility issues. Belonging to the matrix metalloproteinase protein family, MMP-1 is an interstitial collagenase that degrades collagen and may be involved in cell migration, cell proliferation, the pro-inflammatory effect, and cancer progression. Hence, it can serve as a disease protein biomarker and thus be useful in early diagnosis. Herein, epitopes of MMP-1 were identified by screening its crystal structure. To identify possible epitopes for imprinting, MMP-1 was cleaved in silico with trypsin, pepsin at pH = 1.3, and pepsin at pH > 2.0 using Peptide Cutter, generating peptide fragments containing 8 to 12 amino acids. Five criteria were applied to select the peptides most suitable as potential epitopes for MMP-1. The triphenylamine rhodanine-3-acetic acid (TPARA) functional monomer was synthesized to form a stable pre-polymerization complex with a selected template epitope. The complexed functional monomer was then copolymerized with 3,4-ethoxylenedioxythiophene (EDOT) using potentiodynamic electropolymerization onto indium−tin−oxide (ITO) electrodes. The composition of the molecularly imprinted poly(TPARA-co-EDOT) (MIP) was optimized by maximizing the film's electrical conductivity. Cyclic voltammetry was used to determine MMP-1 concentration in the presence of the Fe(CN)63−/Fe(CN)64− redox probe actuating the "gate effect." A calibration curve was constructed and used to determine the usable concentration range and the limit of detection as ca. 0.001 to 10.0 pg/mL and 0.2 fg/mL MMP-1, respectively. Finally, the MMP-1 concentration in the A549 human lung (carcinoma) culture medium was measured, and this determination accuracy was confirmed using an ELISA assay.

Synthesis and application of a "plastic antibody" in electrochemical microfluidic platform for oxytocin determination.

By means of molecular imprinting of a conducting polymer, molecular cavities selective for oxytocin nonapeptide, an autism biomarker, were designed. Embedding of the oxytocin template, and then its extracting from the molecularly imprinted polymer (MIP) was confirmed by the XPS analysis. AFM imaging of the MIP film surface indicated changes in mechanical properties of the film after template extraction. The MIP synthetic receptor was deposited by potentiodynamic electropolymerization as a thin film on an Au film electrode in an electrochemical miniaturized microfluidic cell. The use of this cell allowed to shorten analysis time and to decrease the sample volume. The linear dynamic concentration range extended from 0.06 to 1mM with the limit of detection of 60µM (S/N = 3). Advantageously, sensitivity of the diagnostic microfluidic platform devised for oxytocin determination in both synthetic serum samples and in aqueous solutions was similar and, moreover, it was selective to common interferences, such as oxytocin analogs and potential metabolites.

Molecularly imprinted polymers for separating and sensing of macromolecular compounds and microorganisms.

The present review article focuses on gathering, summarizing, and critically evaluating the results of the last decade on separating and sensing macromolecular compounds and microorganisms with the use of molecularly imprinted polymer (MIP) synthetic receptors. Macromolecules play an important role in biology and are termed that way to contrast them from micromolecules. The former are large and complex molecules with relatively high molecular weights. The article mainly considers chemical sensing of deoxyribonucleic acids (DNAs), proteins and protein fragments as well as sugars and oligosaccharides. Moreover, it briefly discusses fabrication of chemosensors for determination of bacteria and viruses that can ultimately be considered as extremely large macromolecules.

Early diagnosis of fungal infections using piezomicrogravimetric and electric chemosensors based on polymers molecularly imprinted with d-arabitol.

An elevated concentration of d-arabitol in urine, especially compared to that of l-arabitol or creatinine, is indicative of a fungal infection. For that purpose, we devised, fabricated, and tested chemical sensors determining d-arabitol. These chemosensors comprised the quartz crystal resonator (QCR) or extended-gate field-effect transistor (EG-FET) transducers integrated with molecularly imprinted polymer (MIP) film recognition units. To this end, we successfully applied a covalent approach to molecular imprinting, which involved formation of weak reversible covalent bonds between vicinal hydroxyl groups of arabitol and boronic acid substituents of the bithiophene functional monomer used. The MIP films were synthesized and simultaneously deposited on gold electrodes of quartz crystal resonators (Au-QCRs) or Au-glass slides by oxidative potentiodynamic electropolymerization. With the QCR and EG-FET chemosensors, the d-arabitol concentration was determined under flow-injection analysis and stagnant-solution binding conditions, respectively. Selectivity with respect to common interferences, and l-arabitol in particular, of the devised chemosensors was superior. Limits of detection and linear dynamic concentration ranges of the QCR and EG-FET chemosensors were 0.15 mM and 0.15 to 1.25 mM as well as 0.12 mM and 0.12 to 1.00 mM, respectively, being lower than the d-arabitol concentrations in urine of patients with invasive candidiasis (>220 µM). Therefore, the devised chemosensors are suitable for early diagnosis of fungal infections caused by Candida sp. yeasts.