Recognition Reagents Research Articles

Determination of β-Agonists in Porcine Urine by Molecularly Imprinted Polymer Based Chemiluminescence

Isoprenaline was used as the template to synthesize a molecularly imprinted polymer that was able to simultaneously recognize ten β-agonists. The simulation showed that the three-dimensional conformation of the template was the main factor responsible for the polymer’s recognition. The polymer particles were coated in the wells of a conventional microplate as the recognition reagent to prepare a chemiluminescence sensor. The light signal was initiated with a highly effective bis(2,4,6-trichlorophenyl) oxalate-H2O2-imidazole system. The assay contained only one sample-loading step followed by immediate data acquisition, so one measurement was complete within 12 min. The sensor was used to determine the ten analytes in porcine urine. The results showed that the senor achieved ultrahigh sensitivity, with limits of detection from 2.0 to 7.0 pg/mL, high recoveries from 78.6% to 99.4%, and satisfactory recycle performance up to seven times. This is the first study reporting a molecularly imprinted polymer based microtiter chemiluminescence sensor for the determination of β-agonists.

Determination of Tetracyclines in Milk with a Molecularly Imprinted Polymer-Based Microtiter Chemiluminescence Sensor

A molecularly imprinted polymer (MIP) capable of recognizing five tetracyclines using minocycline as the template was synthesized for the first time. The MIP was employed as the recognition reagent to prepare a chemiluminescence sensor on a conventional microtiter plate. The light signal was initiated using the highly efficient bis(2,4,6-trichlorophenyl) oxalate-hydrogen peroxide-imidazole chemiluminescence system. After optimization of several appropriate factors, the sensor was employed to determine five tetracyclines in milk. The developed assay contained only one sample-loading step, so each measurement was completed within 12 min. The limits of detection for these analytes were in the range from 0.5 to 2.0 pg/mL, while the recoveries from the fortified milk samples were between 78.1 and 105%. In addition, the sensor was shown to be reusable for up to four measurements. Hence, this sensor has been demonstrated to be a simple, rapid, sensitive, and durable tool for the determination of tetracyclines in animal-derived food.

In Vitro Selection of a DNA Aptamer by Cell-SELEX as a Molecular Probe for Cervical Cancer Recognition and Imaging.

Aptamers have become the most promising recognition reagents in terms of early diagnosis and effective treatment of cancers. In this study, using cervical cancer as a model, we have identified a DNA aptamer specifically binding to cervical cancer cells with high affinity using the cell-SELEX (systematic evolution of ligands by exponential enrichment) method, in which a negative selection was carried out using normal epithelial cells as control. The binding abilities of 6 selected truncated aptamers were determined by laser confocal fluorescence microscopy and flow cytometry, while most of them only recognize the target cells and do not bind the control cells, and the aptamer C-9S with 51-mer shows the best binding affinity to Ca Ski cells (target cells) with a dissociation constant value of 19.3 ± 2.9nM. Moreover, at physiological temperature, C-9S remains its specific recognition capability to Ca Ski cells as well. Meanwhile, C-9S shows a similar binding ability to another cervical cancer cells (HeLa). Therefore, on the basis of its excellent targeting properties and inherent functional versatility of aptamer, C-9S holds great potential to be a molecular probe for early detection, in vivo imaging, and targeted delivery for further researches in cancer.

Detection of chloramphenicol in chicken, pork and fish with a molecularly imprinted polymer-based microtiter chemiluminescence method

ABSTRACTIn this study, 4-nitrotoluene (NT) was used as dummy template to synthesize a molecularly imprinted polymer that was highly specific for chloramphenicol. The polymer was coated in the wells of 96-well microplates as recognition reagent to develop a chemiluminescence method. The analyte solution and an enzyme-labelled hapten were added into the wells to perform competition, and the light signal was induced with a highly efficient luminol-H2O2-4-(imidazol-1-yl)phenol system. Then, the optimized method was used to determine chloramphenicol in meat (chicken, pork and fish), and the limit of detection (LOD) was 5.0 pg g−1. Furthermore, the polymer-coated plate could be reused four times, and one test could be finished within 20 min. The recoveries from the standard fortified blank meat samples were in the range of 71.5–94.4%. Therefore, this method could be used as a useful tool for routine screening the residue of chloramphenicol in meat samples.

Chemical probes for spatially resolved measurement of active enzymes in single cells.

Ongoing advances in chemical proteomic methods have facilitated detection and quantification of enzymatic activity, a highly informative parameter that is not captured in protein abundance measurements. However, some biological questions remain unanswered, since current gel- or LC-MS/MS-based detection methods suffer from limitations stemming from sample homogenization, signal-averaging, and an inherent bias toward abundant proteins. To address these shortcomings, we recently developed an activity-based proximity ligation (ADPL) platform to capture and quantify enzyme activity on the level of single cells, with high intra- and intercellular spatial resolution. In this chapter, we briefly discuss the rationale behind the ADPL platform, the design transition from the initial "sandwich-complex" workflow to the optimized, "direct conjugate" ADPL method, and conclude with detailed protocols for each. We also describe our novel use of the homo-bifunctional linker, disuccinimidyl suberate (DSS), to conjugate proteins and oligonucleotides, thus generating the necessary antibody-oligonucleotide recognition reagents for ADPL. Finally, we demonstrate the utility of ADPL to characterize enzyme activity from cytosol to nucleus, and specifically detect enzyme activity using "direct conjugate" ADPL.

Preparation of a chemiluminescence sensor for multi-detection of benzimidazoles in meat based on molecularly imprinted polymer

In this study, a molecularly imprinted polymer capable of recognizing 8 benzimidazoles was first synthesized. The computation simulation showed that the shape and size of used template were the main factors influencing its recognition ability. Then the polymer was used as recognition reagent to prepare a chemiluminescence sensor on conventional 96-well microplate. The sample solution and a HRP-labeled hapten were added into the microplate wells to perform competitive binding, and the light signal was initiated with 4-(imidazol-1-yl)phenol enhanced luminol-H2O2 system. The optimized sensor was used to determine the residues of 8 benzimidazoles in mutton and beef. Result showed that the sensor achieved ultrahigh sensitivity (limits of detection of 1.5–21 pg/mL), rapid assay process (18 min) and satisfactory recovery (65.8%–91.2%). Furthermore, this sensor could be reused for 4 times. Therefore, this sensor could be used as a rapid, simple, sensitive and durable tool for screening the residual benzimidazoles in meat.

A molecularly imprinted polymer based chemiluminescence array sensor for one-step determination of phenothiazines and benzodiazepines in pig urine.

The residues of phenothiazines and benzodiazepines in foods of animal origin are dangerous to consumers. For inspection of their abuses, this study for the first time reported on the use of a chemiluminescence array sensor for the simultaneous determination of four phenothiazines and five benzodiazepines in pig urine. Two molecularly imprinted polymers were coated in different wells of a conventional 96-well microtiter plate as the recognition reagents. After sample loading, the absorbed analytes were initiated directly by using an imidazole enhanced bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide system to emit light. The assay process consisted of only one sample-loading step prior to data acquisition, so one test was finished within 10min. The limits of detection for the nine drugs in the pig urine were in a range of 0.1 to 0.6pg/mL, and the recoveries from the fortified blank urine samples were in a range of 80.3 to 95%. Furthermore, the sensor could be reused six times. Therefore, this sensor could be used as a simple, rapid, sensitive and reusable tool for routine screening for residues of phenothiazines and benzodiazepines in pig urine.

Development of Cellulosic Paper-Based Test Strips for Mercury(II) Determination in Aqueous Solution.

Titration method (dropping-on method) was introduced as an efficient approach for determining the mercury ion (Hg2+) concentration in aqueous solution by using fabricated cellulosic paper-based test strips. In this study, dithizone used as a recognition reagent was physically loaded on cellulosic paper-based test strips for Hg2+ selective recognition. The sensing mechanism was established on the spectral absorption rate of the coordination compound that was formed by dithizone and Hg2+ under strong acidic conditions. The calibration curve was obtained by the absorbency of Hg2+-dithizone complexes from different Hg2+ concentration solutions, and the correlation coefficient (R2) reached 0.9971. The detection range of the test trip for Hg2+ was obtained at 0.1 μg/mL to 30 μg/mL. Moreover, these superior cellulosic paper-based test strips have a rapid color-forming time (1.5 min) and low volume demand (3.7 μL samples at 0.0127 g/L dithizone recognition concentration). This portable paper-based test strip can give potential applications for field screening or on-site semiquantitative analysis.

A receptor-based chemiluminescence enzyme linked immunosorbent assay for determination of tetracyclines in milk

This study for the first time reported a receptor based chemiluminescence immunoassay for analysis of tetracyclines. During the experiments, the gene of TetR protein was transformed into Rosetta-gami(DE3) to express a receptor, and its recognition mechanism for 5 tetracyclines was studied by using molecular docking technique. Results showed that hydrophobic interaction and Pi-Pi bond were the main intermolecular forces responsible for TetR-tetracyclines binding, and the D ring was the main receptor binding position in tetracyclines molecules. Then the receptor was used as recognition reagent to develop a direct competitive enzyme linked immunosorbent assay for determination of the 5 drugs in milk, and the light signal was induced with 4-(imidazol-1-yl)phenol enhanced luminol-H2O2 system. The limits of detection for the 5 drugs in milk were in the range of 5–16 pg/mL, and the recoveries from the standards fortified blank milk were in the range of 71.7%–95.8%. Therefore, this method could be used as a simple, rapid, and ultra-sensitive tool to monitor the residues of tetracyclines in milk.

Development and characterization of newly engineered chemosensor with intracellular monitoring potentialities and lowest detection of toxic elements

The widespread existence of potentially toxic heavy elements have become one of the serious environmental concerns around the globe. Thus, it is highly important to develop efficient and selective chemical sensors to detect such potentially toxic elements. Herein, we developed a rhodamine-2-amino-5-bromopyrimidine based chemosensor (RBP) for the selective detection of toxic elements using Hg2+ and Cr3+ as model elements in combination with other competitive ions such as Al3+, Ag+, Ca2+, Ba2+, Cd2+, Fe2+, Fe3+, Li+, K+, Mn2+, Mg2+, Cu2+, Na+, Ni2+, Pb2+and Zn2+. The newly developed RBP displayed good visibility, high sensitivity, excellent selectivity, high binding ability and low limit of detection in CH3CN/HEPES buffer (1 mM 3:2 v/v, pH 7.3) as a medium. The binding constants of RBP for Hg2+ and Cr3+ were 1.09 × 107 M−1 and 8.3 × 104 M−1, respectively. As compared to earlier reported studies, the lowest detection limits (LOD), i.e., 1.6 μM for Hg2+ and 4.9 μM for Cr3+ were recorded. RBP also show reversible binding affinity with Hg2+ and Cr3+ in the presence of ethylenediaminetetraacetate (EDTA). The binding mode between metal ions and RBP were further investigated by using density functional theory (DFT) calculations, which support the experimental findings very well. More importantly, RBP can be prepared as a test paper kit to detect the concentration of Hg2+ and Cr3+ ions by changing the color of the paper visible to naked eyes and is potential for the practical infield application. In addition, the results obtained from confocal microscopy revealed that the probe is cell permeable with low cytotoxicity and can be employed as a bio-imaging reagent for intracellular recognition of Hg2+ and Cr3+ ratification in human breast cancer cells MCF-7.

Molecularly imprinted polymer based microtiter chemiluminescence array for determination of phenothiazines and benzodiazepines in pork

In this study, a molecularly imprinted polymer based chemiluminescence array capable of simultaneous determining phenothiazines and benzodiazepines was first reported. Two polymers were coated in different wells of the conventional 96-well microtiter plate as the recognition reagents, and the added analytes competed with a horseradish peroxidase-labeled bi-hapten conjugate to bind the recognition reagents. The light signal was induced by using a highly effective luminol-H2O2-IMP system. The assay procedure consisted of only one sample-loading step prior to data acquisition. Then, the array was used to determine 4 phenothiazines and 5 benzodiazepines in pork simultaneously. The limits of detection for the 9 drugs were in a range of 0.001–0.01 ng/mL, and the recoveries from the fortified blank pork were in a range of 63.5%–94.1%. Furthermore, the array could be reused for 8 times. The detection results for some real pork samples were consistent with an ultra performance liquid chromatography method.

Aptamer-based photoelectrochemical biosensor for antibiotic detection using ferrocene modified DNA as both aptamer and electron donor

Graphite-like carbon nitride (g-C3N4) nanosheet is a new type of photoactive semiconducting material with promising application in photoelectrochemical (PEC) biosensor. In the present work, a simple and selective PEC biosensor was fabricated using g-C3N4 nanosheets as photoactive material coupled with capture-release strategy. DNA aptamer labeled with ferrocene (DNA-Fc) was served as both recognition reagent of antibiotic chloramphenicol and electron donor of PEC biosensor. Based on the decreased affinity of aptamer to g-C3N4 nanosheets causing by the formation of aptamer-chloramphenicol complex, DNA-Fc could be released from g-C3N4 nanosheets modified ITO surface, resulting a decreased PEC response. The developed method showed a wide linear range from 1 pM to 100 nM and a low detection limit of 0.22 pM (3σ). The fabricated PEC biosensor also illustrated good detection specificity and can be applied to detect chloramphenicol residue in water samples with recoveries ranging from 94.5% to 107.3%.

Chiral imines prepared from 1-(2-aminoalkyl)aziridines as novel chiral shifts reagents for efficient recognition of acids

Optically pure, chiral imines synthesized from the corresponding aldehydes and 1-(2-aminoalkyl)aziridines in good chemical yields, have been assessed as an NMR chiral shift reagents for effective discrimination of the signals of some acids (mandelic acid and its derivatives and N-protected amino acid). The title compounds have proven to be very useful for the determination of enantiomeric purity and absolute configuration of the aforementioned acid derivatives.

Photoelectrochemical apta-biosensor for zeatin detection based on graphene quantum dots improved photoactivity of graphite-like carbon nitride and streptavidin induced signal inhibition

Zeatin is a kind of typical cytokinin, which plays important roles in regulating the proliferation and differentiation of plant cells, and widely distributed in various plant tissues. Herein, a simple and sensitive photoelectrochemical method was developed for zeatin detection using graphite-like carbon nitride (g-C3N4) as photoactive material, graphene quantum dots as photoactivity improvement reagent, gold nanoparticles as immobilization substrate for DNA probe, DNA biotin labeled aptamer as zeatin recognition and streptavidin capture element, and streptavidin as PEC response inhibitor factor. In the presence of zeatin, DNA aptamer can bind with zeatin to form conjugate, which make aptamer releasing from electrode surface and decrease the capture amount of streptavidin, causing the increase of the PEC response. Under optimal conditions, the biosensor showed wide linear range from 0.1 to 100nM and low detection limit of 0.031nM (3σ). The developed method also presented good selectivity, even discriminating zeatin analogues. In addition, the proposed detection strategy can be further applied to detect zeatin in complex biological matrix, revealing great potential in practical applicability. This work might provide an excellent platform for zeatin or other phytohormone detection using aptamer as recognition reagent.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Imprinting of Microorganisms for Biosensor Applications.

There is a growing need for selective recognition of microorganisms in complex samples due to the rapidly emerging importance of detecting them in various matrices. Most of the conventional methods used to identify microorganisms are time-consuming, laborious and expensive. In recent years, many efforts have been put forth to develop alternative methods for the detection of microorganisms. These methods include use of various components such as silica nanoparticles, microfluidics, liquid crystals, carbon nanotubes which could be integrated with sensor technology in order to detect microorganisms. In many of these publications antibodies were used as recognition elements by means of specific interactions between the target cell and the binding site of the antibody for the purpose of cell recognition and detection. Even though natural antibodies have high selectivity and sensitivity, they have limited stability and tend to denature in conditions outside the physiological range. Among different approaches, biomimetic materials having superior properties have been used in creating artificial systems. Molecular imprinting is a well suited technique serving the purpose to develop highly selective sensing devices. Molecularly imprinted polymers defined as artificial recognition elements are of growing interest for applications in several sectors of life science involving the investigations on detecting molecules of specific interest. These polymers have attractive properties such as high bio-recognition capability, mechanical and chemical stability, easy preparation and low cost which make them superior over natural recognition reagents. This review summarizes the recent advances in the detection and quantification of microorganisms by emphasizing the molecular imprinting technology and its applications in the development of sensor strategies.

Fluorometric determination and imaging of glutathione based on a thiol-triggered inner filter effect on the fluorescence of carbon dots

The authors describe a sensitive method for determination of glutathione (GSH) that is based on a thiol-triggered inner filter effect on the fluorescence of N-doped carbon dots (N-doped CDs). N-doped CDs with a quantum yield as high as 31% were prepared by a one-pot procedure, and 5,5′-dithiobis-(2-nitrobenzoic acid) was employed as a reagent for GSH recognition. The reaction product (5-thio-2-nitrobenzoic acid; TNB) acts as an absorber of the 410-nm light used to photo-excite the N-doped CDs. Hence, the fluorescence of N-doped CDs (peaking at 510 nm) is reduced with increasing concentrations of GSH. As little as 30 nM of GSH can be detected by this method. The approach was successfully applied to (a) food analysis, (b) an investigation of an oxidative stress model, and (c) to live cells imaging. The method does not require the surface of N-doped CDs to be chemically modified, and a linkage between receptor and fluorophore is not needed. In our perception, the method may become a viable tool for the detection and imaging of thiols.

Pairing Alpaca and Llama-Derived Single Domain Antibodies to Enhance Immunoassays for Ricin

Previously, our group isolated and evaluated anti-ricin single domain antibodies (sdAbs) derived from llamas, engineered them to further increase their thermal stability, and utilized them for the development of sensitive immunoassays. In work focused on the development of therapeutics, Vance et al. 2013 described anti-ricin sdAbs derived from alpacas. Herein, we evaluated the utility of selected alpaca-derived anti-ricin sdAbs for detection applications, and engineered an alpaca-derived sdAb to increase its melting temperature, providing a highly thermal stable reagent for use in ricin detection. Four of the alpaca-derived anti-ricin A-chain sdAbs were produced and characterized. All four bound to epitopes that overlapped with our previously described llama sdAbs. One alpaca sdAb, F6, was found to possess both a high melting temperature (73 °C) and to work optimally with a thermally stable llama anti-ricin sdAb in sandwich assays for ricin detection. We employed a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond to further enhance the thermal stability of F6 to 85 °C. It is advantageous to have a choice of recognition reagents when developing assays. This work resulted in defining an additional pair of highly thermal stable sdAbs for the sensitive detection of ricin.

Peptide Antibodies in Clinical Laboratory Diagnostics.

Peptide antibodies, with their high specificities and affinities, are invaluable reagents for peptide and protein recognition in biological specimens. Depending on the application and the assay, in which the peptide antibody is to used, several factors influence successful antibody production, including peptide selection and antibody screening. Peptide antibodies have been used in clinical laboratory diagnostics with great success for decades, primarily because they can be produced to multiple targets, recognizing native wildtype proteins, denatured proteins, and newly generated epitopes. Especially mutation-specific peptide antibodies have become important as diagnostic tools in the detection of various cancers. In addition to their use as diagnostic tools in malignant and premalignant conditions, peptide antibodies are applied in all other areas of clinical laboratory diagnostics, including endocrinology, hematology, neurodegenerative diseases, cardiovascular diseases, infectious diseases, and amyloidoses.

Electrochemical immunosensor for N6-methyladenosine detection in human cell lines based on biotin-streptavidin system and silver-SiO2 signal amplification

N6-methyladenosine (m6A), a kind of RNA methylation form and important epigenetic event, plays crucial roles in many biological progresses. Thus it is essential to quantitatively detect m6A in complicated biological samples. Herein, a simple and sensitive electrochemical method was developed for m6A detection using N6-methyladenosine-5′-triphosphate (m6ATP) as detection target molecule. In this detection strategy, anti-m6A antibody was selected as m6A recognition and capture reagent, silver nanoparticles and amine-PEG3-biotin functionalized SiO2 nanospheres (Ag@SiO2) was prepared and used as signal amplification label, and phos-tag-biotin played a vital role of “bridge” to link m6ATP and Ag@SiO2 through the two forms of specific interaction between phosphate group of m6ATP and phos-tag, biotin and streptavidin, respectively. Under the optimal experimental conditions, the immunosensor presented a wide linear range from 0.2 to 500nM and a low detection limit of 0.078nM (S/N=3). The reproducibility and specificity were acceptable. Moreover, the developed method was also validated for detect m6A content in human cell lines. Importantly, this detection strategy provides a promising immunodetection platform for ribonucleotides and deoxyribonucleotides with the advantages of simplicity, low-costing, specificity and sensitivity.