Bifunctionalized nanobioprobe based rapid color-shift assay for typhoid targeting Vi capsular polysaccharide.

Typhoid fever is an acute illness caused by Salmonella Typhi and the current diagnostic gap leads to inaccurate, over-diagnosis of typhoid leading to excessive use of antibiotics. Herein, to address the challenges we describe a new rapid color-shift assay based on a novel bifunctional nanobioprobe (Vi-AgNP probe) that is functionalized with specific biomarker Vi polysaccharide and also has the co-presence of Ag as urease inhibitor. The immunoreactions between the Vi with specific antibodies (Abs) present in typhoid patient sample forms a shielding barrier over Vi-AgNP probe rendering the urease to be active, generating colored output. Vi polysaccharide coating on the AgNP was visualized using HRTEM. TEM was performed to get insight into shielding barrier formation by the Abs. MST (microscale thermophoresis) data showed less binding Kd of 7.43 µM in presence of Abs whereas probe with urease showed efficient binding with Kd 437 nM. The assay was validated using 53 human sera samples and proven effective with 100% sensitivity. The assay showed relative standard deviation (RSD) of 4.3% estimated using rabbit anti-Vi Abs. The entire procedure could be completed within 15 min. Unlike lateral flow based assays, our assay does not require multiple combination of Abs for detection. The assay format was also found compatible in paper strip test that provides promising opportunities to develop low-cost on-spot assay for clinical diagnostics.

Conformational switching of aptamer biointerfacing graphene-gold nanohybrid for ultrasensitive label-free sensing of cardiac Troponin I.

The development of hybrid biofunctionalized nanomaterials has emerged as an attractive substitute for development of advanced biosensing platforms with superior synergistic properties. Herein, we report a label-free ultrasensitive electrochemical aptasensor comprising nanohybrid of graphene oxide (GO) and aptamer conjugated gold nanoparticles (GNP-A) for detection of cardiac biomarker Troponin I (TnI). The GNP-A are homogenously arranged by self-assembly on GO sheet to construct nanohybrid (GO@GNP-A) onto which the biomarker protein is analysed. TnI interactions at the aptamer biointerfaced nanohybrid surface causes electrochemical signal enhancement probed by using a redox active molecule. The consecutive increase in current signal is strongly attributed to conformational switching of aptamer and charge neutralization at the interface induced by TnI binding. The sensitivity of the nanohybrid aptasensor platform was found to be 0.001 pg/mL. The study has been further substantiated in Acute Myocardial Infarction (AMI) clinical samples for usage towards early, sensitive and efficient point-of-care detection of TnI.

Mycobacterium tuberculosis exploits MPT64 to generate myeloid-derived suppressor cells to evade the immune system.

Mycobacterium tuberculosis (Mtb) is a smart and successful pathogen since it can persist in the intimidating environment of the host by taming and tuning the immune system. Mtb releases MPT64 (Rv1980c) protein in high amounts in patients with active tuberculosis (TB). Consequently, we were curious to decipher the role of MPT64 on the differentiating dendritic cells (DCs) and its relation to evading the immune system. We observed that pre-exposure of differentiating DCs to MPT64 (DCMPT64) transformed them into a phenotype of myeloid-derived suppressor cells (MDSCs). DCMPT64 expressed a high level of immunosuppressive molecules PD-L1, TIM-3, nitric oxide (NO), arginase 1, IDO-1, IL-10 and TGF-ß, but inhibited the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-12. DCMPT64 chemotaxis function was diminished due to the reduced expression of CCR7. DCMPT64 promoted the generation of regulatory T cells (Tregs) but inhibited the differentiation of Th1 cells and Th17 cells. Further, high lipid and methylglyoxal content, and reduced glucose consumption by DCMPT64, rendered them metabolically quiescent and consequently, reduced DCMPT64 ability to phagocytose Mtb and provided a safer shelter for the intracellular survival of the mycobacterium. The mechanism identified in impairing the function of DCMPT64 was through the increased production and accumulation of methylglyoxal. Hence, for the first time, we demonstrate the novel role of MPT64 in promoting the generation of MDSCs to favor Mtb survival and escape its destruction by the immune system.

Targeting dendritic cells with TLR-2 ligand-coated nanoparticles loaded with Mycobacterium tuberculosis epitope induce antituberculosis immunity.

Novel vaccination strategies are crucial to efficiently control tuberculosis, as proposed by the World Health Organization under its flagship program "End TB Strategy." However, the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), particularly in those coinfected with HIV-AIDS, constitutes a major impediment to achieving this goal. We report here a novel vaccination strategy that involves synthesizing a formulation of an immunodominant peptide derived from the Acr1 protein of Mtb. This nanoformulation in addition displayed on the surface a toll-like receptor-2 ligand to offer to target dendritic cells (DCs). Our results showed an efficient uptake of such a concoction by DCs in a predominantly toll-like receptor-2-dependent pathway. These DCs produced elevated levels of nitric oxide, proinflammatory cytokines interleukin-6, interleukin-12, and tumor necrosis factor-α, and upregulated the surface expression of major histocompatibility complex class II molecules as well as costimulatory molecules such as CD80 and CD86. Animals injected with such a vaccine mounted a significantly higher response of effector and memory Th1 cells and Th17 cells. Furthermore, we noticed a reduction in the bacterial load in the lungs of animals challenged with aerosolized live Mtb. Therefore, our findings indicated that the described vaccine triggered protective anti-Mtb immunity to control the tuberculosis infection.

Site-directed dual bioprobes inducing single-step nano-sandwich assay for the detection of cardiac troponin I.

Bioreceptor functionalized metallic nano-colloids have been identified as effective nanobioprobes to realize the detection of an analyte based on a common phenomenon of salt-induced aggregation. In marked contrast to this, we describe a nano-sandwich assay integrating the novel match-pair of aptamer and peptide functionalized gold nanoparticles. The site-directed biomolecular interaction of high affinity aptamer and peptide bioreceptors directed towards distinct sites of cardiac biomarker troponin I; this was found to form a nano-sandwich assay in a peculiar manner. The gold nanoconjugates interact with specific and distant regions of troponin I to result in collision of probes upon target identification. In the presence of TnI, both nanobioprobes bind at their respective sites forming a nano-sandwich pair providing a visual color change from red to blue. Thus, the presence of target TnI itself causes instant agglomeration in just a single-step without addition of any external aggregator. The assay imparts 100% specificity and 90% sensitivity in a dynamic concentration range of 0.1-500 ng/mL troponin I with detection limit as low as 0.084 ng/mL. The applicability of the assay has been validated in clinical samples of acute myocardial infarction patients thus establishing a promising point-of-care detection of TnI.

Deciphering the Antibacterial Role of Peptide From Bacillus subtilis subsp. spizizenii Ba49 Against Staphylococcus aureus.

An increase in antibiotic resistance has led to escalating the need for the development of alternate therapy. Antimicrobial peptides (AMPs) are at the forefront of replacing conventional antibiotics, showing slower development of drug resistance, antibiofilm activity, and the ability to modulate the host immune response. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that jeopardize most conventional antibiotics are known to be involved in severe respiratory tract, bloodstream, urinary tract, soft tissue, and skin infections. Among them, S. aureus is an insidious microbe and developed resistance against conventional antibiotics. In the present study, an AMP (named as peptide-Ba49) isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa (the common onion) exhibited strong antibacterial efficacy against S. aureus ATCC 25923. The mode of action of this peptide-Ba49 on S. aureus was deciphered through various sensitive probes, i.e., DiSC3 (5) and H2DCFDA, suggesting the peptide-Ba49 to be acting upon through change in membrane potential and by triggering the production of reactive oxygen species (ROS). This induced disruption of the cell membrane was further supported by morphological studies using scanning electron microscopy (SEM). Investigations on a possible post-antibiotic effect (PAE) of peptide-Ba49 showed prolonged PAE against S. aureus. Furthermore, the peptide-Ba49 prevented the formation of S. aureus biofilm at low concentration and showed its potential to degrade the mature biofilm of S. aureus. The peptide-Ba49 also exhibited intracellular killing potential against S. aureus ATCC 25923 in the macrophage cells, and moreover, peptide-Ba49 was found to bolster the fibroblast cell migration in the scratch assay at low concentration, exhibiting a wound healing efficacy of this peptide. These studies demonstrated that peptide-Ba49 isolated from the strain B. subtilis subsp. spizizenii could be a therapeutic candidate to combat the pathogenic S. aureus infections.

Colorimetric and electrochemical detection of pathogens in water using silver ions as a unique probe.

The manuscript highlights the efficacy of silver ions to act as a unique probe for the detection of bacterial contamination in water samples. The bacterial cell membrane adherence property of the silver ions was employed to develop two different bacterial detection assays employing colorimetric and electrochemical techniques. In one of the schemes, silver ion was used directly as a detector of bacteria in a colorimetric assay format, and in the other scheme surface-functionalized antibodies were used as a primary capture for specific detection of Salmonella enterica serovar Typhi. The colorimetric detection is based on silver-induced inhibition of urease activity and silver ion utilization by bacteria for the rapid screening of enteric pathogens in water. The specific detection of bacteria uses an antibody-based electrochemical method that employs silver as an electrochemical probe. The ability of silver to act as an electrochemical probe was investigated by employing Anodic Stripping Voltammetry (ASV) for targeted detection of Salmonella Typhi. For further insights into the developed assays, inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) studies were performed. The sensitivity of the developed assay was found to be 100 cfu mL-1 for colorimetric and 10 cfu mL-1 for electrochemical assay respectively.

Gold nanoparticle triggered siloxane formation for polymerization based amplification in enzyme free visual immunoassay.

Herein, we report a new signal amplification scheme for quantitative biochemical analysis based on gold nanoparticle (GNPs) catalyzed polymerization of transparent silane solution to milky white and turbid siloxane. Using immunoassay as a proof of concept, GNP labeled immunoprobe was used to bind captured antigen and catalyse the polymerization reaction allowing sensitive biochemical investigation. The polymerization reaction was optimized for standard 96 well polystyrene microtiter plates and we discovered that sodium lactate acts as an enhancer in the polymerization reaction as it reduces detection time to merely 30 min. The sensing strategy was applied to detection and quantification of Salmonella Typhimurium in water and egg samples and the platform showed excellent visibly quantifiable analytical response up to 100 cells mL-1. Furthermore, clinical utility and potential of the method was validated by detecting Vi capsular polysaccharide (Vi antigen) responsible for typhoidal Salmonellosis in human serum in sandwich format with a detection limit of 1 ng mL-1. The method serves as the first report towards nanoparticle triggered polymerization for development of rapid and low cost quantitative biochemical assay.

Rapid and sensitive colorimetric detection of pathogens based on silver-urease interactions.

Herein, we demonstrate a facile and economic approach for colorimetric detection of microbial pathogens in drinking water, employing silver-urease interactions. In the presence of harmful pathogens, receptor coated silver nanoparticles (AgNPs) preferentially bind to the bacterial surface and urease catalytically elevates the pH of the solution, which is sensed by a pH responsive chromogenic dye. The assay demarcates bacterial contamination levels up to 102 cells mL-1 in a field-friendly method.

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.

Graphene-gated biochip for the detection of cardiac marker Troponin I.

We report lithium ion intercalation mediated efficient exfoliation of graphite to form monolithic graphene sheets which have subsequently been investigated for the development of highly sensitive label-free electrochemical detection platform for cardiac biomarker, Troponin I (cTnI). The spectroscopic and morphological analysis demonstrated the formation of defect free graphene sheets which were successfully employed to fabricate an inter-digited microdevice in a drain-source configuration on a silicon biochip. The graphene gated biochip functionalized with anti-cTnI antibodies used in label free detection of cTnI which exhibited an excellent sensitivity in the picogram range (~1 pg mL(-1)) for cTnI without the use of any enzymatic amplification that promises its potential applicability for bio-molecular detection in clinical diagnosis.

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.

CdTe nanobioprobe based optoelectrochemical immunodetection of diabetic marker HbA1c.

Highly luminescent water soluble CdTe quantum dots (QDs) were synthesized and conjugated with anti-HbA1c antibody to generate specific nanobioprobe. A sandwich immunoassay model was employed using capture HbA1c antibody as a specific receptor molecule and the QD-labeled secondary antibody as a dual (fluorescence cum electrochemical) tracer to quantify the concentration of HbA1c. A linear increase in current was observed for HbA1c analytical standards with a R(2) value of 0.990 and coefficient of variance ~5%. A comparison between HPLC and dual immunoassay for clinical samples showed a correlation coefficient of 89% and 96% for fluorescence and electrochemical detection methods respectively. The QD-based immunoassay shows great promise for rapid reproducible and cost effective analysis of HbA1c in clinical samples.

Amine functionalized graphene oxide/CNT nanocomposite for ultrasensitive electrochemical detection of trinitrotoluene.

Binding of electron-deficient trinitrotoluene (TNT) to the electron rich amine groups on a substrate form specific charge-transfer Jackson-Meisenheimer (JM) complex. In the present work, we report formation of specific JM complex on amine functionalized reduced graphene oxide/carbon nanotubes- (a-rGO/CNT) nanocomposite leading to sensitive detection of TNT. The CNT were dispersed using graphene oxide that provides excellent dispersion by attaching to CNT through its hydrophobic domains and solubilizes through the available OH and COOH groups on screen printed electrode (SPE). The GO was reduced electrochemically to form reduced graphene that remarkably increases electrochemical properties owing to the intercalation of high aspect CNT on graphene flakes as shown by TEM micrograph. The surface amine functionalization of dropcasted and rGO/CNT was carried out using a bi-functional cross linker ethylenediamine. The extent of amine functionalization on modified electrodes was confirmed using energy dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS) and confocal microscopy. The FTIR and Raman spectra further suggested the formation of JM complex between amine functionalized electrodes and TNT leading to a shift in peak intensity together with peak broadening. The a-rGO/CNT nanocomposite prepared electrode surface leads to ultra-trace detection of TNT upto 0.01 ppb with good reproducibility (n=3). The a-rGO/CNT sensing platform could be an alternate for sensitive detection of TNT explosive for various security and environmental applications.

Bio-functionalized graphene-graphene oxide nanocomposite based electrochemical immunosensing.

We report a novel in-situ electrochemical synthesis approach for the formation of functionalized graphene-graphene oxide (fG-GO) nanocomposite on screen-printed electrodes (SPE). Electrochemically controlled nanocomposite film formation was studied by transmission electron microscopy (TEM) and Raman spectroscopy. Further insight into the nanocomposite has been accomplished by the Fourier transformed infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) spectroscopy. Configured as a highly responsive screen-printed immunosensor, the fG-GO nanocomposite on SPE exhibits electrical and chemical synergies of the nano-hybrid functional construct by combining good electronic properties of functionalized graphene (fG) and the facile chemical functionality of graphene oxide (GO) for compatible bio-interface development using specific anti-diuron antibody. The enhanced electrical properties of nanocomposite biofilm demonstrated a significant increase in electrochemical signal response in a competitive inhibition immunoassay format for diuron detection, promising its potential applicability for ultra-sensitive detection of range of target analytes.

Enhancing electrochemical detection on graphene oxide-CNT nanostructured electrodes using magneto-nanobioprobes.

Graphene and related materials have come to the forefront of research in electrochemical sensors during recent years due to the promising properties of these nanomaterials. Further applications of these nanomaterials have been hampered by insufficient sensitivity offered by these nanohybrids for the type of molecules requiring lower detection ranges. Here, we report a signal amplification strategy based on magneto-electrochemical immunoassay which combines the advantages of carbon nanotube and reduced graphene oxide together with electrochemical bursting of magnetic nanoparticles into a large number of metal ions. Sensitive detection was achieved by precisely designing the nanohybrid and correlating the available metal ions with analyte concentration. We confirmed the ultrahigh sensitivity of this method for a new generation herbicide diuron and its analogues up to sub-picomolar concentration in standard water samples. The novel immune-detection platform showed the excellent potential applicability in rapid and sensitive screening of environmental pollutants or toxins in samples.

Rapid extraction and quantitative detection of the herbicide diuron in surface water by a hapten-functionalized carbon nanotubes based electrochemical analyzer.

A solid phase extraction micro-cartridge containing a non-polar polystyrene absorbent matrix was coupled with an electrochemical immunoassay analyzer (EIA) and used for the ultra-sensitive detection of the phenyl urea herbicide diuron in real samples. The EIA was fabricated by using carboxylated carbon nanotubes (CNTs) functionalized with a hapten molecule (an amine functionalized diuron derivative). Screen printed electrodes (SPE) were modified with these haptenized CNTs and specific in-house generated anti diuron antibodies were used for bio-interface development. The immunodetection was realized in a competitive electrochemical immunoassay format using alkaline phosphatase labeled secondary anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of an electrochemically active product, 1-naphthol, which was monitored by using differential pulse voltammetry (DPV). The assay exhibited excellent sensitivity and specificity having a dynamic response range of 0.01 pg mL(-1) to 10 µg mL(-1) for diuron with a limit of detection of around 0.1 pg mL(-1) (n = 3) in standard water samples. The micro-cartridge coupled hapten-CNTs modified SPE provided an effective and efficient electrochemical immunoassay for the real-time monitoring of pesticides samples with a very high degree of sensitivity.

Self-assembly and sensor response of photosynthetic reaction centers on screen-printed electrodes.

Photosynthetic reaction centers were immobilized onto gold screen-printed electrodes (Au-SPEs) using a self-assembled monolayer (SAM) of mercaptopropionic acid (MPA) which was deliberately defective in order to achieve effective mediator transfer to the electrodes. The pure Photosystem II (PS II) cores from spinach immobilize onto the electrodes very efficiently but fair badly in terms of photocurrent response (measured using duroquinone as the redox mediator). The cruder preparation of PS II known as BBY particles performs significantly better under the same experimental conditions and shows a photocurrent response of 20-35 nA (depending on preparation) per screen-printed electrode surface (12.5mm(2)). The data was corroborated using AFM, showing that in the case of BBY particles a defective biolayer is indeed formed, with grooves spanning the whole thickness of the layer enhancing the possibility of mass transfer to the electrodes and enabling biosensing. In comparison, the PS II core layer showed ultra-dense organization, with additional formation of aggregates on top of the single protein layer, thus blocking mediator access to the electrodes and/or binding sites. The defective monolayer biosensor with BBY particles was successfully applied for the detection of photosynthesis inhibitors, demonstrating that the inhibitor binding site remained accessible to both the inhibitor and the external redox mediator. Biosensing was demonstrated using picric acid and atrazine. The detection limits were 1.15 nM for atrazine and 157 nM for picric acid.

A novel disposable electrochemical immunosensor for phenyl urea herbicide diuron.

A disposable electrochemical immunosensor has been developed for the determination of phenyl urea herbicide-diuron using a low cost laser ablated gold electrodes (LC-LAGE) fabricated on polystyrene substrate. The electrodes were electrochemically deposited with prussian blue-gold nanoparticle (PB-GNP) film, and a competitive inhibition immunoassay was performed on LC-LAGE by using a specific hapten-protein conjugate. The binding of available diuron specific antibody on conjugate coated electrode was detected using alkaline phosphatase rabbit anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of electrochemically active product, 1-naphthol, which was monitored using square wave voltammetry technique. The assay exhibited an excellent sensitivity and specificity showing the dynamic response range between 1 ppt and 10 ppm for diuron with detection limit around 1 ppt. This study provides insight into development of a rapid and high-throughput screening of pesticides in environmental samples at a very low cost.

New insights for using self-assembly materials to improve the detection stability in label-free DNA-chip and immuno-sensors.

This paper examines reliable advancements in low-cost DNA- and immuno-chips. Capacitance detection was successfully chosen to develop label-free bio-chips. Probe immobilization was rigorously investigated in order to obtain reliable capacitance measurements. Protein probes immobilized by using usual alkanethiols or thiolated ssDNA probes directly immobilized on gold do not allow sufficient stable capacitance measurements. New alkanethiols improved with ethylene-glycol function are shown in this paper to be more suitable materials for capacitive bio-chip development. Atomic Force Microscopy, Quartz Crystal Microbalance, and Capacitance Measurements were used to demonstrate that ethylene-glycol alkanethiols allow high time stability, smaller errors in detection, and improved ideal behaviour of the sensing surfaces. Measured capacitance is in the range of 8-11 nF/mm(2) for antibody layers and close to 6 nF/mm(2) for DNA probes. It is in the range of 10-12 nF/mm(2) and of 4-6 nF/mm(2) for antigen and DNA detection, respectively. The percentage error in detection is highly improved and it is in the range of 11-37% and of 0,23-0,82% for antigen and DNA, respectively. The reproducibility is also improved and it is close to 0,44% for single spot measurements on ethylene-glycol alkanethiols. A molecular theory attributing these improvements to water molecules strongly coordinated by ethylene-glycol functional groups and to solution ions not entering into probe films is finally proposed.