Biolayer interferometry-SELEX for Shiga toxin antigenic-peptide aptamers & detection via chitosan-WSe2 aptasensor.

We report biolayer interferometry based in-vitro selection technique (BLI-SELEX) for fishing out specific aptamers against E. coli Shiga toxin subtypes viz., stx1 & stx2 via epitopic peptides. BLI-SELEX is a one-step technique for rapidly generating aptamers against protein biomarkers in a microtiter plate format, obliterating the need for multiple enrichment rounds to harvest high-affinity aptamers as in conventional SELEX. Two unique aptamers selected against stx1 & stx2 with picomolar Kd (~47 pM & ~29 pM, respectively) were successfully used to fabricate voltammetric diagnostic assay via immobilization onto chitosan exfoliated 2D tungsten diselenide (WSe2) nanosheet platform. These aptamers modified nanosensors showed high sensitivity of ~ 5.0 µA ng-1 mL, a dynamic response range from 50 pg mL-1 to 100 ng mL-1, with a detection limit of 44.5 pg mL-1 & 41.3 pg mL-1 for stx subtypes, respectively and showed low cross-reactivity in spiked urine, serum and milk samples. The synergistic effect of selective aptamers & high sensitivity imparted by 2D transition metal dichalcogenide (TMD) highlights the superior potential of a fabricated nanosensor for bacterial toxin detection.

Electrochemical aptasensor using boron-carbon nanorods decorated by nickel nanoparticles for detection of E. coli O157:H7.

The development of a label-free impedimetric aptasensor is reported for rapid and sensitive detection of Escherichia coli O157:H7 employing boron-carbon nanorods decorated by nickel nanoparticles (BC-Ni) nanostructured platform. These highly electroactive BC-Ni nanorods were synthesized to increase the sensitivity of the sensor surface and subsequently functionalized with a specific anti-E. coli O157:H7 aptamer (Kd = 69 nM) as bio-recognition moiety. This fully characterized high-affinity DNA aptamer against the bacteria was selected using a facile microtiter plate-based cell-SELEX methodology. The fabricated electrochemical aptasensor is demonstrated to detect E. coli O157:H7 selectively with a detection limit of 10 cfu and a dynamic detection range of 100 to 105 cfu in water, juice, and fecal samples. Graphical abstract.

Graphitic carbon nitride QDs impregnated biocompatible agarose cartridge for removal of heavy metals from contaminated water samples.

Highly fluorescent, water-stable graphitic carbon nitride quantum dots (gCN QDs) synthesized by microwave assisted solvo-thermal technique and characterized via optical spectroscopy, XRD, HR-TEM, Fluorescence spectroscopy, FT-IR and Raman spectroscopy. Synthesized gCN were used for the removal of mercury ions from polluted water samples in a microcartridge format. Density functional theory (DFT) calculations revealed a possible interaction of mercury atoms, and embedment of mercury atom onto synthesized gCN surface lead to moderate structural distortion, reduced band gap and altered dielectric response. Experimentally, the excitation dependent fluorescence of QDs is highly compromised in presence of mercuric (Hg2+) and other ions, validating the theoretical findings, and establishing their use as metal sensor probes. Hg2+ binding ability with gCN QDs was further utilized in developing bioinspired micro-cartridge via covalent conjugation to Agarose microbeads. Micro-cartridge can remove heavy metal contamination from polluted water with a binding efficiency of 24.63 mg HgCl2 for 10 mg of Agarose-gCN conjugate.

Trace detection of some nitro-explosives using thermal mediated immunochemical defragmented method.

A new immunoassay format using thermally induced defragmentation of some nitro-explosives with a high degree of selectivity is reported. Specific antibodies against three widely used explosives, 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN) were generated by designing suitable haptens using geometry optimization modules. These in-house generated antibodies were used in a newly developed thermal mediated immunochemical biosensing technique which involves the binding of specific antibodies to respective nitro-explosives on a microtiter strip, resulting in the formation of specific immunocomplex. Heating the specific immuno-complex formed on microtiter wells resulted in thermal lysis of nitro-explosives to generate nitrite ions. These ions react with Griess reagent to form a colored chromophore which correlates the concentration of individual explosive in the sample. The present work fulfills the need for an improved explosive detecting system that is highly specific and capable of quickly determining the presence of nitrate containing explosives from a mixture pool.

Aptamer functionalized MoS2-rGO nanocomposite based biosensor for the detection of Vi antigen.

We report a novel aptamer functionalized MoS2-rGO based electrochemical method for Vi polysaccharide antigen mediated detection of enteric fever. Herein, highly selective anti-Vi aptamers were screened from a pool of oligonucleotides using a microtitre based SELEX approach and characterized for its specificity and stability. The MoS2-rGO nanocomposite was synthesized using a liquid assisted exfoliation by taking optimum ratio of MoS2 and rGO. The nanocomposite presented synergistic effect owing to easy biomolecular functionalization and enhanced conductivity. The screened anti-Vi aptamers were embedded on the MoS2-rGO nanocomposite via thiol linkage to give a stable biointerface. The developed aptasensor was characterized and further evaluated for its performance with different concentrations of Vi antigen using ferrocene labeled boronic acid as an electroactive probe. The aptasensor responded linearly in the range between 0.1 ng mL-1 to 1000 ng mL-1with a detection limit of 100 pg mL-1, and did not show any cross-reactivity with other bacterial polysaccharides indicating high specificity. The applicability of the developed aptasensor was further validated in urine and sera specimens spiked with Vi antigen.

Plasmonic DNA hotspots made from tungsten disulfide nanosheets and gold nanoparticles for ultrasensitive aptamer-based SERS detection of myoglobin.

A nanohybrid mediated SERS substrate was prepared by in-situ synthesis and assembly of gold nanoparticles (AuNPs) on exfoliated nanosheets of tungsten disulfide (WS2) to form plasmonic hotspots. The nanohybrid surface was functionalized with specific aptamers which imparted high selectivity for the cardiac marker myoglobin (Mb). The fabricated aptasensor was read by SERS using a 532 nm laser and demonstrated significant signal enhancement, and this allowed Mb to be determined in the 10 f. mL-1 to 0.1 µg mL-1 concentration range. The study presents an approach to synergistically exploit the unique chemical and electromagnetic properties of both WS2 and AuNPs for many-fold enhancement of SERS signals. Graphical abstract Schematic presentation of a nanohybrid-mediated SERS substrate prepared by in-situ assembly of gold nanoparticles (AuNPs) reduced on exfoliated nanosheets of tungsten disulfide (WS2) to form plasmonic hot spots. Specific aptamers immobilized on the SERS surface impart high sensitivity and selectivity for the cardiac marker myoglobin (Mb).

Bridged Rebar Graphene functionalized aptasensor for pathogenic E. coli O78:K80:H11 detection.

We report a novel fabrication method of functionalised Bridged Rebar Graphene (BRG) onto newly designed nanostructured aptasensor for label free impedimetric sensing of pathogenic bacteria E. coli O78:K80:H11. The chemical facilitated unscrolling of MWCNT and subsequent bridging with terephthalaldehyde (TPA) to form 3D-hierarchical BRG nanoconstruct exhibited synergistic effect by combining enhanced electrical properties and facile chemical functionality for stable bio-interface. The bacteria-DNA interactions were captured on BRG nanostructured electrode by using specific anti-E.coli DNA aptamer (Kd~ 14nM), screened by new in-situ developed SELEX method using phenylboronic acid on microtitre plate. The developed nanostructured aptasensor demonstrated a low detection limit and sensitivity of ~ 101cfu/mL towards E. coli O78:K80:H11 with a dynamic response range from 101 to 106cfu/mL in water, juice and milk samples.

Nanostructured Aptamer-Functionalized Black Phosphorus Sensing Platform for Label-Free Detection of Myoglobin, a Cardiovascular Disease Biomarker.

We report the electrochemical detection of the redox active cardiac biomarker myoglobin (Mb) using aptamer-functionalized black phosphorus nanostructured electrodes by measuring direct electron transfer. The as-synthesized few-layer black phosphorus nanosheets have been functionalized with poly-l-lysine (PLL) to facilitate binding with generated anti-Mb DNA aptamers on nanostructured electrodes. This aptasensor platform has a record-low detection limit (∼0.524 pg mL(-1)) and sensitivity (36 µA pg(-1) mL cm(-2)) toward Mb with a dynamic response range from 1 pg mL(-1) to 16 µg mL(-1) for Mb in serum samples. This strategy opens up avenues to bedside technologies for multiplexed diagnosis of cardiovascular diseases in complex human samples.

A New Electrolytic Synthesis Method for Few-Layered MoS2 Nanosheets and Their Robust Biointerfacing with Reduced Antibodies.

We report an efficient method for the synthesis of few-layered MoS2 nanosheets and demonstrate their application in the label-free detection of the prostate-specific antigen (PSA) cancer marker. As a novel strategy, the electro-dissolution of molybdenum metal sheets in the presence of Na(+) and S(2-) ions led to the formation of Na(+) intercalated MoS2. Further exfoliation by ultrasonication yielded the desired formation of few-layered MoS2 nanosheets. After comprehensive characterization, the synthesized MoS2 nanosheets were channeled in a field-effect transistor (FET) microdevice. Chemically reduced anti-PSA antibodies were immobilized on the MoS2 channel above the FET microdevice to construct a specific PSA immunosensor. The antibodies were deliberately reduced to expose the hinge-region disulfide bonds. This approach offered a robust and site-directed immunosensing device through biointerfacing of the sulfhydryl groups (-SH) in the reduced antibody with the surface S atoms of MoS2. This device was validated as an effective immunosensor with a low detection limit (10(-5) ng/mL) over a wide linear detection range (10(-5) to 75 ng/mL).

Graphene-CNT nanohybrid aptasensor for label free detection of cardiac biomarker myoglobin.

We report a label free electrochemical detection of cardiac bio-marker myoglobin (Mb) on aptamer functionalized rGO/CNT nanostructured electrodes by measuring its direct electron transfer (DET). Configured as a highly responsive aptasensor, the newly developed biosensing platform exhibits synergistic effect of the nano-hybrid functional construct by combining good electrical properties and the facile chemical functionality of nanohybrid for the compatible bio-interface development. The specific anti-Mb aptamer was generated by five iterative SELEX (Systematic evolution of ligands by exponential enrichment) rounds, showing high senstivity (KD ~65 pM). The aptamer functionalized rGO/CNT nanostructured electrodes demonstrated a significant increase in signal response with a detection limit of ~0.34 ng/mL in the dynamic response range between 1 ng/mL and 4 µg/mL for Mb. The newly developed DET assay format presents a promising candidate in point-of-care diagnosis for routine screening of Mb in patient's samples.

Biofunctionalized rebar graphene (f-RG) for label-free detection of cardiac marker troponin I.

One-step microwave-assisted unscrolling of carbon nanotubes to form functionalized rebar graphene (f-RG) is reported. The well-characterized f-RG on an interdigitated electrode biochip in a FET configuration showed enhanced electronic properties, as demonstrated with I-V characteristics. The developed device was biofunctionalized with specific anti-cTnI antibodies exhibiting a shift of threshold voltage from -2.15 V to -0.5 V and decrease in electron mobility from 3.609 × 10(4) to 8.877 × 10(3) cm(2) V(-1) s(-1). The new sensing strategy holds great promise for its applicability in diagnostics exhibiting high sensitivity (∼ 1 pg/mL) and specificity toward cardiac marker (cTnI).

Hybrid aptamer-antibody linked fluorescence resonance energy transfer based detection of trinitrotoluene.

Combining synthetic macromolecules and biomolecular recognition units are promising in developing novel diagnostic and analysis techniques for detecting environmental and/or clinically important substances. Fluorescence resonance energy transfer (FRET) apta-immunosensor for explosive detection is reported using 2,4,6-trinitrotoluene (TNT) specific aptamer and antibodies tagged with respective FRET pair dyes in a sandwich immunoassay format. FITC-labeled aptamer was used as a binder molecule in the newly developed apta-immunoassay format where the recognition element was specific anti-TNT antibody labeled with rhodamine isothiocyanate. The newly developed sensing platform showed excellent sensitivity with a detection limit of the order of 0.4 nM presenting a promising candidate for routine screening of TNT in samples.