An aptasensor for rapid and sensitive detection of estrogen receptor alpha in human breast cancer.

The analysis of estrogen receptor (ER) expression in breast carcinomas plays a crucial role in determining the endocrine responsiveness of tumors for systemic adjuvant therapy. Conventionally, the ER levels in breast carcinomas had been detected using the dextran-coated charcoal assay and radioimmunoassay, which are now substituted with safer and economic antibody-based assays such as immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Despite a gold (Au) standard method, the IHC has been criticized for factors such as tissue fixation, antibody selection, and threshold staining for result interpretation that could falsify test accuracy and reproducibility. The quest for alternative methods of ER quantification in tissue samples paved the way for aptamer-based diagnostics. Previously, we have isolated a DNA aptamer against human ER alpha (ERα) using an in vitro evolution system. In this study, we developed an electrochemical sensor using the 76-nucleotide DNA ERα- aptamer for rapid, precise, and cost-effective detection of ERα expression in human breast cancer patients. The aptasensor was constructed by covalently immobilizing the thiolated ERα- aptamer onto a screen-printed Au electrode. Construction of aptasensors was confirmed through atomic force microscopy and differential pulse voltammetry measurements. A detection limit of 0.001 ng/ml was calculated for full-length ERα (66.2 kDa) in a detection time of 10 min. Analysis of the cancerous breast tissue samples using the ELISA and aptasensor methods enabled distinctive classification of samples into the categories of ER -ve, weak ER +ve, and strong ER +ve samples. The current change of this aptasensor lies within 5% after a storage of 60 days at 4°C. Further studies on a reasonably large sample size are required to realize the clinical potential of the sensor.

Moderate reagent mixing on an orbital shaker reduces the incubation time of enzyme-linked immunosorbent assay.

Rapid diagnostic tests can be developed using ELISA for detection of diseases in emergency conditions. Conventional ELISA takes 1-2 days, making it unsuitable for rapid diagnostics. Here, we report the effect of reagents mixing via shaking or vortexing on the assay timing of ELISA. A 48-min protocol of ELISA involving 12-min incubations with reagent mixing at 750 rpm for every step was optimized. Contrary to this, time-optimized control ELISA performed without mixing produced similar results in 8 h, leaving a time gain of 7 h using the developed protocol. Collectively, the findings suggest the development of ELISA-based rapid diagnostics.

Exploring the flexible chemistry of 4-fluoro-3-nitrophenyl azide for biomolecule immobilization and bioconjugation.

Bioconjugation and functionalization of polymer surfaces are two major tasks in materials chemistry which are accomplished using a variety of coupling agents. Immobilization of biomolecules onto polymer surfaces and the construction of bioconjugates are essential requirements of many biochemical assays and chemical syntheses. Different linkers with a variety of functional groups are used for these purposes. Among them, the benzophenones, aryldiazirines, and arylazides represent the most commonly used photolinker to produce the desired chemical linkage upon their photo-irradiation. In this review, we describe the versatile applications of 4-fluoro-3-nitrophenyl azide, one of the oldest photolinkers used for photoaffinity labeling in the late 1960s. Surprisingly, this photolinker, historically known as 1-fluoro-2-nitro-4-azidobenzene (FNAB), has remained unexplored for a long time because of apprehension that FNAB forms ring-expanded dehydroazepine as a major product and hence cannot activate an inert polymer. The first evidence of photochemical activation of an inert surface by FNAB through nitrene insertion reaction was reported in 2001, and the FNAB-activated surface was found to conjugate a biomolecule without any catalyst, reagent, or modification. FNAB has distinct advantages over perfluorophenyl azide derivatives, which are contemporary nitrene-generating photolinkers, because of its simple, single-step preparation and ease of thermochemical and photochemical reactions with versatile polymers and biomolecules. Covering these aspects, the present review highlights the flexible chemistry of FNAB and its applications in the field of surface engineering, immobilization of biomolecules such as antibodies, enzymes, cells, carbohydrates, oligonucleotides, and DNA aptamers, and rapid diagnostics. Graphical Abstract An overview of the FNAB-engineered activated polymer surfaces for covalent ligation of versatile biomolecules.

Microwave-Assisted Rapid Enzymatic Synthesis of Nucleic Acids.

Herein we report microwave-induced enhancement of the reactions catalyzed by Escherichia coli DNA polymerase I and avian myeloblastosis virus-reverse transcriptase. The reactions induced by microwaves result in a highly selective synthesis of nucleic acids in 10-50 seconds. In contrast, same reactions failed to give desired reaction products when carried out in the same time periods, but without microwave irradiation. Each of the reactions was carried out for different duration of microwave exposure time to find the optimum reaction time. The products produced by the respective enzyme upon microwave irradiation of the reaction mixtures were identical to that produced by the conventional procedures. As the microwave-assisted reactions are rapid, microwave could be a useful alternative to the conventional and time consuming procedures of enzymatic synthesis of nucleic acids.

Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer.

An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (Ka = 1.55±0.298×108 M(-1); ΔH = 4.32×104±801.1 cal/mol; ΔS = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.

Ultraminiaturized assay for rapid, low cost detection and quantification of clinical and biochemical samples.

Herein, we report a simple, sensitive, rapid and low-cost ultraminiaturized assay technique for quantitative detection of 1 µl of clinical or biochemical sample on a novel ultraminiaturized assay plate (UAP). UAP is prepared by making tiny cavities on a polypropylene sheet. As UAP cannot immobilize a biomolecule through absorption, we have activated the tiny cavities of UAP by 1-fluoro-2-nitro-4-azidobenzene in a photochemical reaction. Activated UAP (AUAP) can covalently immobilize any biomolecule having an active nucleophilic group such as amino group. Efficacy of AUAP is demonstrated by detecting human IgE, antibody of hepatitis C virus core antigen and oligonucleotides. Quantification is performed by capturing the image of the colored assay solution and digitally quantifying the image by color saturation without using costly NanoDrop spectrophotometer. Image - based detection of human IgE and an oligonucleotide shows an excellent correlation with absorbance - based assay (recorded in a NanoDrop spectrophotometer); it is validated by Pearson's product-moment correlation with correlation coefficient of r = 0.9545088 and r = 0.9947444 respectively. AUAP is further checked by detecting hepatitis C virus Ab where strong correlation of color saturation with absorbance with respect to concentration is observed. Ultraminiaturized assay successfully detects target oligonucleotides by perfectly hybridizing with their respective complementary oligonucleotide probes but not with a random oligonucleotide. Ultraminiaturized assay technique has substantially reduced the requirement of reagents by 100 times and assay timing by 50 times making it a potential alternative to conventional method.

In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules.

Aptamers, the chemical-antibody substitute to conventional antibodies, are primarily discovered through SELEX technology involving multi-round selections and enrichment. Circumventing conventional methodology, here we report an in silico selection of aptamers to estrogen receptor alpha (ERα) using RNA analogs of human estrogen response elements (EREs). The inverted repeat nature of ERE and the ability to form stable hairpins were used as criteria to obtain aptamer-alike sequences. Near-native RNA analogs of selected single stranded EREs were modelled and their likelihood to emerge as ERα aptamer was examined using AutoDock Vina, HADDOCK and PatchDock docking. These in silico predictions were validated by measuring the thermodynamic parameters of ERα -RNA interactions using isothermal titration calorimetry. Based on the in silico and in vitro results, we selected a candidate RNA (ERaptR4; 5'-GGGGUCAAGGUGACCCC-3') having a binding constant (Ka) of 1.02 ± 0.1 × 10(8) M(-1) as an ERα-aptamer. Target-specificity of the selected ERaptR4 aptamer was confirmed through cytochemistry and solid-phase immunoassays. Furthermore, stability analyses identified ERaptR4 resistant to serum and RNase A degradation in presence of ERα. Taken together, an efficient ERα-RNA aptamer is identified using a non-SELEX procedure of aptamer selection. The high-affinity and specificity can be utilized in detection of ERα in breast cancer and related diseases.

Development of a label-free gold nanoparticle-based colorimetric aptasensor for detection of human estrogen receptor alpha.

The increasing demand for easily available and low-cost diagnostics has fuelled the development of aptasensors as platforms for rapid, sensitive, and point-of-care testing of target analytes. Recently, gold nanoparticle (AuNP)-based aptasensors have attracted wide recognition owing to their color transition properties which allow real-time rapid sensing of targets. In this study, we utilized the color transition property of aptamer-functionalized AuNPs to detect and quantify estrogen receptor alpha (ERα), a key biomarker protein in breast cancer. We found that the coating of AuNPs with unmodified ERα-RNA aptamer (GGGGUCAAGGUGACCCC) makes them resistant to salt-induced aggregation. However, addition of ERα to the aptamer-protected AuNPs results in their spontaneous aggregation as evident from a color transition from wine red to deep blue. On the basis of this, we developed an ERα aptasensor, with limits of detection and quantification of 0.64 and 2.16 ng/mL, respectively; the aptasensor can efficiently detect and quantify ERα in a working range of 10 ng/mL-5µg/mL protein. Validation of the aptasensor on cellular extracts of ERα-positive MCF-7 and ERα-deficient MDA-MB-231 breast cancer cells showed a target-selective response in ERα-positive samples but not in cellular extracts of ERα-deficient breast cancer cells. Further, the small size and simple fabrication chemistry of aptamers provide an additional benefit to make the ERα aptasensor a potentially useful and cost-effective tool in point-of-care analyses of ERα.

Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A.

Screening and identification of a DNA aptamer to concanavalin A and its application in food analysis.

Herein, a novel aptamer that targets concanavalin A (Con A), a plant lectin, is isolated using systematic evolution of ligands by an exponential enrichment (SELEX) technique. Nine rounds of SELEX screening over an agarose spin column have resulted in enrichment of eight sequences having high affinity to Con A. The highest affinity sequence was selected as a potent aptamer and characterized it in detail. The evolved Con A aptamer (Con A-aptabody) is a 41 nt ssDNA that binds the Con A specifically with a dissociation constant of 172.7 ± 29.7 nM. In silico analyses predict the Con A-aptabody to form G-quadruplex due to its G-rich sequence (GGAAGGCGGAGGG). A detection method developed using Con A-aptabody is found to have a detection range of 10-750 ng/mL with limits of detection and quantification being 13.22 and 44.09 ng/mL, respectively. The utility of the method is demonstrated by analyzing jack bean (Canavalia ensiformis), kidney bean (Phaseolus vulgaris), wheat (Triticum spp.), mung bean (Vigna radiata), and lentil (Lens culinaris) for their Con A contents. Hence, the developed Con A-aptabody provides a useful substitute to Con A-antibody for food analysis and related applications.

Microwave-mediated enzymatic modifications of DNA.

Here we report microwave-induced specific cleavage, ligation, dephosphorylation, and phosphorylation of nucleic acids catalyzed by restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase. The microwave-mediated method has dramatically reduced the reaction time to 20 to 50s. In control experiments, the same reactions failed to give the desired reaction products when carried out in the same time periods but without microwave irradiation. Because the microwave method is rapid, it could be a useful alternative to the time-consuming conventional procedure for enzymatic modification of DNA.

Femtogram detection of horseradish peroxidase by a common desktop scanner.

We report an image-based detection of horseradish peroxidase (HRP) by different color spaces. The results show excellent correlation between color saturation and absorbance (Pearson correlation coefficient; 0.9868) with respect to HRP. The present method can detect 185 and 46.45 fg/ml of HRP using o-phenylenediamine dihydrochloride and 3,3',5,5'-tetramethylbenzidine as chromogenic substrates respectively.

Image-based detection of oligonucleotides--a low cost alternative to spectrophotometric or fluorometric methods.

Herein, we report a sensitive and low cost image-based (photocolorimetric) method for the detection of oligonucleotides on an activated polypropylene microtest plate (APPµTP). The assay was developed on the APPµTP by covalently immobilising 20-mer amino-modified oligonucleotides. Biotin-tagged complementary target sequences were then hybridised with the immobilised oligonucleotides. Colour was developed by streptavidin-HRP conjugate and the image of the coloured assay solution was taken by a desktop scanner and analysed using colour saturation. The developed method was analysed for its detection limit, accuracy, sensitivity and interference. The linearity range was found to be 1.7-170 ng mL(-1) while the lower limit of detection and limit of quantification were 1.7 and 5.6 ng mL(-1) respectively. The method showed comparable sensitivity to fluorometric methods, and was found to be correlated to fluorescence (R(2) = 0.8081, p-value < 0.0001) and absorbance (R(2) = 0.9394, p-value < 0.0001)-based quantification. It discriminates mismatched base sequences from perfectly matched sequences efficiently. Validation of the method was carried out by detecting por A DNA of Neisseria meningitidis in bacterial meningitis samples. The por A-specific probe having a 6-carbon spacer at its 5'-NH2 terminus was immobilised covalently to the APPµTP and hybridised with different samples of biotinylated single-stranded por A DNA.

Identification of novel adhesins of M. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis.

Pathogenic bacteria interacting with eukaryotic host express adhesins on their surface. These adhesins aid in bacterial attachment to the host cell receptors during colonization. A few adhesins such as Heparin binding hemagglutinin adhesin (HBHA), Apa, Malate Synthase of M. tuberculosis have been identified using specific experimental interaction models based on the biological knowledge of the pathogen. In the present work, we carried out computational screening for adhesins of M. tuberculosis. We used an integrated computational approach using SPAAN for predicting adhesins, PSORTb, SubLoc and LocTree for extracellular localization, and BLAST for verifying non-similarity to human proteins. These steps are among the first of reverse vaccinology. Multiple claims and attacks from different algorithms were processed through argumentative approach. Additional filtration criteria included selection for proteins with low molecular weights and absence of literature reports. We examined binding potential of the selected proteins using an image based ELISA. The protein Rv2599 (membrane protein) binds to human fibronectin, laminin and collagen. Rv3717 (N-acetylmuramoyl-L-alanine amidase) and Rv0309 (L,D-transpeptidase) bind to fibronectin and laminin. We report Rv2599 (membrane protein), Rv0309 and Rv3717 as novel adhesins of M. tuberculosis H37Rv. Our results expand the number of known adhesins of M. tuberculosis and suggest their regulated expression in different stages.

Image-based ELISA on an activated polypropylene microtest plate--a spectrophotometer-free low cost assay technique.

In this communication, we report ELISA technique on an activated polypropylene microtest plate (APPµTP) as an illustrative example of a low cost diagnostic assay. Activated test zone in APPµTP binds a capture biomolecule through covalent linkage thereby, eliminating non-specific binding often prevalent in absorption based techniques. Efficacy of APPµTP is demonstrated by detecting human immunoglobulin G (IgG), human immunoglobulin E (IgE) and Aspergillus fumigatus antibody in patient's sera. Detection is done by taking the image of the assay solution by a desktop scanner and analyzing the color of the image. Human IgE quantification by color saturation in the image-based assay shows excellent correlation with absorbance-based assay (Pearson correlation coefficient, r=0.992). Significance of the relationship is seen from its p value which is 4.087e-11. Performance of APPµTP is also checked with respect to microtiter plate and paper-based ELISA. APPµTP can quantify an analyte as precisely as in microtiter plate with insignificant non-specific binding, a necessary prerequisite for ELISA assay. In contrast, paper-ELISA shows high non-specific binding in control sera (false positive). Finally, we have carried out ELISA steps on APPµTP by ultrasound waves on a sonicator bath and the results show that even in 8 min, it can convincingly differentiate a test sample from a control sample. In short, spectrophotometer-free image-based miniaturized ELISA on APPµTP is precise, reliable, rapid, and sensitive and could be a good substitute for conventional immunoassay procedures widely used in clinical and research laboratories.

Characterization of canthaxanthin isomers isolated from a new soil Dietzia sp. and their antioxidant activities.

Canthaxanthin (cx) is a potent antioxidant that is chemically synthesized at the industrial scale and has imperative applications in the cosmetic and feed industries. An orange pigmented mesophilic bacterium, designated as K44, was isolated from soil samples of Kargil, India. Biochemical tests, 16S rRNA gene sequencing, and FAME analysis of the bacterium indicated it to belong in the genus Dietzia and is distinct from human isolates. The strain showed 98% 16S rRNA gene sequence homology with Dietzia maris DSM 43102. High-performance liquid chromatography profile of the pigments isolated from K44 showed two major peaks absorbing at 465.3 and 475 nm. The liquid chromatography-mass spectrometry (LC-MS) analysis of both these peaks revealed their m/z to be 564. The molecular weights, LC-MS/MS fragmentation patterns, and lambdamax of these fractions corresponded to all-trans- (475 nm) and 9-cis-(465.3 nm) cx isomers. The antioxidant activities of cis- and trans-cx isomers isolated from this bacterium were found to differ, where the cis-isomer showed higher free radical, superoxide radical, and reactive oxygen species scavenging activities than the alltrans- isomer, suggesting that 9-cis-cx is more effective as an antioxidant than the all-trans-cx.

Covalent immobilization of ascorbate oxidase onto polycarbonate strip for L-ascorbic acid detection.

Herein, a simple and rapid method is described for detection of L-ascorbic acid by ascorbate oxidase immobilized onto polycarbonate strip pre-activated by 1-fluoro-2-nitro-4-azidobenzene in photochemical reaction. Covalent attachment of ascorbate oxidase was confirmed by XPS studies. The immobilized-ascorbate oxidase shows higher pH, thermal and storage stability in comparison to free enzyme.

Microwave-mediated enzyme-linked immunosorbent assay procedure.

Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), human interleuken 1ß (IL-1ß), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diagnosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters.

Rapid immobilization of enzymes onto solid supports by ultrasound waves.

Herein, we describe a non-conventional method for immobilization of enzymes onto different solid surfaces using ultrasound as a source of energy. When horseradish peroxidase (HRP) was taken on the surface of an activated support and allowed to float on a sonicator bath operating at a frequency of 40 KHz, it readily started binding itself to the surface. Maximum binding was observed in 10 min whereas a control experiment carried out similarly without ultrasound waves showed insignificant immobilization. Ultrasound wave-mediated immobilization is rapid and reproducible and is better suitable for versatile applications in different fields, including fabrication of enzyme-based biosensors or bioreactors.