Revisiting the cortisol reference ranges in humans: the role of demographics.

The current study explores the effect of demographics on serum cortisol expression in a study group of 52 individuals to improve the current serum reference ranges to produce personalized expression profiles consequently increasing clinical confidence in the diagnosis. The serum cortisol concentration was inspected against demographical data like age, sex, and body mass index and showed an association with age and sex. The serum cortisol values also indicated a positive association with chronic illnesses however this finding requires a more focused study for establishment. Additionally, saliva samples are also collected from the same study group at the same time through drool and an absorbent sponge and correlated with serum values to draw an alternative route of serological testing. Salivary cortisol from drool showed a linear correlation with Pearson's correlation coefficients of 0.71 and 0.72 with serum cortisol and with saliva samples collected using a porous sponge respectively. Overall, the study shows the role of demographics in shaping the reference ranges for cortisol, suggesting a path for developing personalized diagnostics. The study also highlights the efficacy of saliva as an alternative to serum cortisol to facilitate cortisol measurement for efficient stress management.

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.

Nanomaterial based aptasensors for clinical and environmental diagnostic applications.

Nanomaterials have been exploited extensively to fabricate various biosensors for clinical diagnostics and food & environmental monitoring. These materials in conjugation with highly specific aptamers (next-gen antibody mimics) have enhanced the selectivity, sensitivity and rapidness of the developed aptasensors for numerous targets ranging from small molecules such as heavy metal ions to complex matrices containing large entities like cells. In this review, we highlight the recent advancements in nanomaterial based aptasensors from the past five years also including the basics of conventionally used detection methodologies that paved the way for futuristic sensing techniques. The aptasensors have been categorised based upon these detection techniques and their modifications viz., colorimetric, fluorometric, Raman spectroscopy, electro-chemiluminescence, voltammetric, impedimetric and mechanical force-based sensing of a multitude of targets are discussed in detail. The bio-interaction of these numerous nanomaterials with the aptameric component and that of the complete aptasensor with the target have been studied in great depth. This review thus acts as a compendium for nanomaterial based aptasensors and their applications in the field of clinical and environmental diagnosis.

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).

Microtitre Plate Based Cell-SELEX Method.

Aptamers have emerged as a novel category in the field of bioreceptors due to their wide applications ranging from biosensing to therapeutics. Several variations of their screening process, called SELEX have been reported which can yield sequences with desired properties needed for their final use. We report a facile microtiter plate-based Cell-SELEX method for a gram-negative bacteria E. coli. The optimized protocol allows the reduction of number of rounds for SELEX by offering higher surface area and longer retention times. In addition, this protocol can be modified for other prokaryotic and eukaryotic cells, and glycan moieties as target for generation of high affinity bio-receptors in a short course of time in-vitro.

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.

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.

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.

Nanobioprobe mediated DNA aptamers for explosive detection.

Specific nucleic acid aptamers using the microtiter plate based modified SELEX method against explosive trinitrotoluene are reported. Efficient partitioning of dsDNA was carried out using streptavidin labeled gold nanoprobes for the selection of specific aptamers. The selected binders having an affinity of ~10(-7) M were used in the newly developed electrochemical aptasensor, exhibiting a detection limit of around 1 ppb for trinitrotoluene.