Upconverting nanoparticle clustering based rapid quantitative detection of tetrahydrocannabinol (THC) on lateral-flow immunoassay.

Cannabis, also known as marijuana, is the most abused psychoactive drug worldwide. Several countries are legalizing the medicinal and recreational use of cannabis. At the same time, stricter laws are being drafted for driving or working under the influence of the drug. Therefore, there is a significant need for rapid point-of-care testing of cannabis in biological fluids. Tetrahydrocannabinol (THC) is the parent psychoactive molecule present in cannabis and is used as a biomarker for cannabis detection. In this work, we developed an upconverting nanoparticle (UCNP) based lateral-flow immunoassay (LFIA) for the point-of-care quantitative detection of THC in oral fluids of drug-impaired personnel. UCNPs convert near-infrared excitation into visible emissions and have superior properties over other fluorescent nanoparticles. We employed a novel signal amplification technique for enhancing the sensitivity of LFIA. Contrary to standard LFIA biosensors, we integrated an additional enhancement pad (EP) between the conjugate pad (CP) and nitrocellulose membrane. UCNPs dually conjugated with THC specific immunoglobulin G and streptavidin (UCNP-IgG-SA) and UCNPs conjugated with biotin (UCNP-biotin) were dried in CP and EP, respectively. UCNP-IgG-SA, upon interacting with THC, flow through the EP and bind with UCNP-biotin, consequently forming bright UCNP clusters on the test and control zones. The test signals were captured after an assay time of 20 minutes. An experimental detection limit of 2 ng mL-1 for THC with a linear detection range of 2-15 ng mL-1 was achieved. The developed LFIA has the potential to be used as a point-of-care detection device for the quantitative detection of THC.

Microchip capillary electrophoresis based electroanalysis of triazine herbicides.

The number of pesticides used in agriculture is increasing steadily, leading to contamination of soil and drinking water. Herein, we present a microfluidic platform to detect the extent of contamination in soil samples. A microchip capillary electrophoresis system with in-channel electrodes was fabricated for label-free electroanalytical detection of triazine herbicides. The sample mixture contained three representative triazines: simazine, atrazine and ametryn. The electropherogram for each individual injection of simazine, atrazine and ametryn showed peaks at 58, 66 and 72 s whereas a mixture of them showed distinct peaks at 59, 67 and 71 s respectively. The technique as such may prove to be a useful qualitative and quantitative tool for the similar environmental pollutants.

Homogeneous electrochemical assay for protein kinase activity.

Herein, we report a homogeneous assay for protein kinase activity using an electrochemistry-based probe. The approach involves a peptide substrate conjugated with a redox tag and the phosphate-specific receptor immobilized on an electrode surface. The peptide substrate phosphorylated by a protein kinase binds to the receptor site of the probe, which results in a redox current under voltammetric measurement. Our method was successfully applied even in the presence of citrated human blood and modified to enable a single-use, chip-based electrochemical assay for kinase activity.

Nonacog Alfa for Prophylaxis and Treatment of Bleeding Episodes in Previously Treated Patients with Moderately Severe or Severe Hemophilia B in India.

Purpose: Hemophilia B is an X-linked congenital bleeding disorder caused by a deficiency of coagulation factor IX (FIX) clotting activity. This study evaluated safety and efficacy of nonacog alfa, a recombinant human blood coagulation FIX replacement product, in males aged 12-65 years with hemophilia B (FIX activity ≤ 2%) with or without inhibitors in India. Methods: In this multicenter, open-label, post-approval phase 4 study, participants were treated for up to 8 weeks, with up to a 4-week screening period and a subsequent post-treatment 28-day safety observation period. Intravenous nonacog alfa 40 IU/kg (range 13-78 IU/kg) was administered at intervals of 3-4 days, in accordance with the approved local product document. Results: A total of 25 participants were enrolled and completed the study. No participants developed FIX inhibitors during the study, experienced treatment-related adverse events (AEs) or serious AEs, or developed a thrombotic event and/or hypersensitivity reaction. No participants experienced bleeding events requiring on-demand treatment with nonacog alfa. Seventeen bleeding episodes (16 spontaneous and 1 traumatic) were reported in 10 participants; all occurred post treatment, with the exception of a minor gum-bleeding event, and were managed without treatment. The mean (SD) annualized total factor consumption (TFC) per patient was 224,582 (75,527) IU; the mean (SD) annualized TFC by weight per patient was 3639 (573) IU/kg. Conclusion: Nonacog alfa was safe and effective for the prevention of hemorrhagic episodes in Indian males with congenital, severe hemophilia B. No participants developed FIX inhibitors, and no new safety signals were reported.

Moroctocog Alfa (AF-CC) for Prophylaxis and Treatment of Bleeding Episodes in Previously Treated Patients with Hemophilia A in India.

Purpose: Hemophilia A is an X-linked congenital disorder, characterized by factor VIII (FVIII) deficiency. Globally, India has the highest population of patients with hemophilia, and there is a clear unmet need for appropriate and effective treatment for this patient population. This multicenter, open-label, post-approval study evaluated the safety and efficacy of moroctocog alfa in patients with moderate or severe congenital hemophilia A in India. Methods: Intravenous moroctocog alfa was administered 30 ± 5 IU/kg 3 times weekly for bleeding prophylaxis, according to the local product document. Participants were treated for up to 8 weeks, with an up to 4-week screening period and a subsequent post-treatment, 28-day safety observation period. Patients continued in the study until at least 24 exposure days or a period of up to 8 weeks on moroctocog alfa. Results: A total of 50 participants were enrolled, and 48 (85.7%) completed the study. No participants developed FVIII inhibitors during the study. The mean (SD) annualized bleeding rate during moroctocog alfa prophylaxis was 0.79 (2.0) with a median (range) of 0.00 (0.0, 6.8). The mean (SD) annualized total factor consumption (TFC) per participant was 287,432 (93,866) IU; the mean (SD) annualized TFC by weight per participant was 4176 (858) IU/kg. Moroctocog alfa was well tolerated with no reported treatment-emergent adverse event-related dose reductions, discontinuations, or serious adverse events. Conclusion: Moroctocog alfa was safe, effective, and well tolerated in Indian participants with congenital moderate to severe hemophilia A. No participant developed FVIII inhibitors during the study.

Capillary electrophoresis microchip for direct amperometric detection of DNA fragments.

Detection and quantitation of nucleic acids have gained much importance in the last couple of decades, especially in the post-human genome project era. Such processes are tedious, time consuming and require expensive reagents and equipment. Therefore, in the present study, we demonstrated a simple process for the separation and analysis of small DNA fragments using capillary electrophoretic amperometric detection on an inexpensive disposable glass microchip. The device used polydimethylsiloxane engraved microchannel and Au/Ti in-channel microelectrodes for sample detection. The DNA fragments were separated under low electric field (20 V/cm) for improved detection sensitivity and to retain the biomolecules in their native conformation. With a low sample requirement (as low as 1 µL) and high reproducibility, the proposed microchip device was successful in resolution and detection of DNA fragments of various lengths.

Microfluidic biosensor for beta-amyloid(1-42) detection using cyclic voltammetry.

Numerous studies have identified beta-amyloid(1-42) protein (Abeta42) in the cerebrospinal fluid as a potential biomarker of Alzheimer's disease. It is of particular interest to establish the diagnosis before reaching the stage of clinical severity. The current methods for studying amyloid detection, however, is often time-consuming, expensive, and labor intensive, making the analytical process very slow. Thus, a critical need exists for an analytical system that would enable the rapid investigation of amyloid formation with a very small amount of amyloidogenic peptides and reagents. In our present work, we report a simple microfluidic biosensor to analyze very small quantities of Abeta42 peptide on gold surface that were modified with Au nano-particles onto the thiol groups of self-assembled 1,6-hexandithiol cross-linkers. The vital advantage of this method includes retaining the bioactivity and environment similar to nature for protein immobilization while using minimal amounts of reagents and highly sensitive detection.

A phase 4 study of the safety of the 13-valent pneumococcal conjugate vaccine in children 6 to 17 years of age in India.

PURPOSE: The 13-valent pneumococcal conjugate vaccine (PCV13) was recently approved in India for the prevention of pneumococcal disease in children aged 6 to 17 years based on global data as well as immunogenicity and safety findings from a phase 3 study. The current phase 4 study in India further evaluated the safety profile of PCV13 in this age group to support the positive benefit-risk profile of PCV13. METHODS: Healthy male and female children aged 6 to 17 years in India were administered a single intramuscular injection of PCV13. Through 7 days after PCV13 administration, local reactions and systemic events were recorded daily by caregivers in an electronic diary. Adverse events (AEs) were collected from the provision of informed consent through 28-42 days postvaccination. RESULTS: One hundred subjects enrolled in and completed the study. After PCV13 vaccination, 73.9% and 57.8% of subjects reported local reactions and systemic events, respectively. The majority of reactogenicity events were mild to moderate in severity, with injection site pain and fatigue the most frequently reported local reaction and systemic event, respectively. Six subjects reported 7 AEs, all of which were considered unrelated to PCV13. One subject reported a serious AE (acute hepatitis), which was considered unrelated to PCV13 and ultimately resolved. No subjects withdrew because of AEs, and there were no deaths. CONCLUSION: PCV13 vaccination was well tolerated with an acceptable safety profile in healthy subjects aged 6 to 17 years in India. This work further supports the safety profile of PCV13 for prevention of pneumococcal disease in this age group in India.

Safety and immunogenicity of a multidose vial formulation of 13-valent pneumococcal conjugate vaccine administered with routine pediatric vaccines in healthy infants in India: A phase 4, randomized, open-label study.

PURPOSE: This phase 4, randomized, open-label, multicenter study in healthy Indian infants and toddlers evaluated the safety, tolerability, and immunogenicity of the 13-valent pneumococcal conjugate vaccine (PCV13) formulated in a multidose vial (MDV) or single prefilled syringe (PFS). METHODS: Healthy Indian infants (6 weeks of age) were randomized 1:1 to receive either PCV13-MDV or PCV13-PFS concomitant with routine pediatric vaccines. Subjects received a single dose of either PCV13-MDV or PCV13-PFS as a 4-dose schedule (infant series: 1 dose at 6, 10, and 14 weeks of age; toddler dose: 12 months of age). Safety was assessed, including local reactions, systemic events, and adverse events (AEs). Immunogenicity 1 month after both the infant series and toddler dose was measured by concentrations of serotype-specific immunoglobulin G (IgG) antibodies and opsonophagocytic activity titers. RESULTS: Rates and severities of local reactions and systemic events up to 7 days after each dose of either PCV13-MDV or PCV13-PFS were generally similar, with the majority being of mild or moderate severity. PCV13-MDV had a safety profile comparable with PCV13-PFS; both groups experienced a similar frequency of AEs. PCV13-MDV elicited immune responses comparable with those induced by PCV13-PFS. Clear boosting of immune responses after the PCV13-MDV toddler dose was observed; ≥96% of subjects showed serotype-specific IgG concentrations at or above the defined thresholds 1 month after the PCV13-MDV toddler dose. CONCLUSIONS: PCV13-MDV was safe, well tolerated, and immunogenic in healthy Indian infants and toddlers when coadministered with routine pediatric vaccinations. Safety and immunogenicity of PCV13-MDV was comparable with PCV13-PFS. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov: NCT03548337.

Phosphorene-gold nanocomposite based microfluidic aptasensor for the detection of okadaic acid.

Okadaic acid (OA) is one of the most prevalent and largely distributed bio-toxin in the world. Consumption of OA results in a series of digestive ailments such as nausea and diarrhea. This study demonstrates the preparation and functioning of an electrochemical microfluidic biochip for the detection of OA. The screen-printed carbon electrode (SPCE) was modified by phosphorene-gold nanocomposite onto which an aptamer specific to OA was immobilized. BP-Au nanocomposites were synthesized by an in-situ, one-step method without the use of a reducing agent. Potassium ferro-ferri cyanide was used as a redox pair to quantify signal strength. To improve reaction time, increase sensitivity and portability, a microfluidic platform was designed and developed. This device comprised of channels identified for specific purposes such as sample mixing and incubation. Overall, the integrated system consisted of a polydimethylsiloxane microfluidic chip housing an aptamer modified SPCE, as a single detection module for Okadaic acid. The nanomaterials and the microfluidic channels prepared were spectroscopically and electrochemically analyzed. Differential pulse voltammograms revealed a detection limit of 8 pM, while a linear range was found between 10 nM-250 nM. Selectivity studies were also performed with spiked mussel samples and other interfering species. This point-of-care device can be deployed to perform on-farm assays in fishing units.

A 2D transition-metal dichalcogenide MoS2 based novel nanocomposite and nanocarrier for multiplex miRNA detection.

Nanoscale MoS2 has attracted extensive attention for sensing due to its superior properties. This study outlines a microfluidic and electrochemical biosensing methodology for the multiplex detection of paratuberculosis-specific miRNAs. Herein, we report the synthesis of MoS2 nanosheets decorated with a copper ferrite (CuFe2O4) nanoparticle composite and molecular probe immobilized MoS2 nanosheets as nanocarriers for the electrochemical detection of miRNAs. Paratuberculosis is a bacterial infection of the intestinal tract of dairy cattle, and is a cause of substantial economic and animal losses all over the world. The designed biosensing electrode was modified with the synthesized MoS2-CuFe2O4 nanocomposites for a highly amplified signal generation. Additionally, selective detection of miRNAs was accomplished by functionalizing the MoS2 nanosheets with a miRNA-specific biotin-tagged thiolated molecular probe and ferrocene thiol. The presence of target miRNA triggered the opening of the molecular probe present on the nanocarriers. The interaction of the molecular probe and miRNA resulted in an increase in the electrochemical signal from ferrocene. The optimized microfluidic biosensor was employed to detect a range of miRNA concentrations from the target analyte. Using square wave voltammetric analysis, a detection limit of 0.48 pM was calculated, with a detection range of 1 pM to 1.5 nM. The application of the biosensor was also assessed by detecting miRNAs in spiked serum and positive clinical samples. The developed nanomaterial enabled biosensor easily discriminated between the target miRNAs and other interfering molecules. The developed microfluidic biosensor has the potential to be used as a point-of-care, miRNA based diagnostic tool for paratuberculosis in dairy cows.

Biosensors for Sustainable Food Engineering: Challenges and Perspectives.

Current food production faces tremendous challenges from growing human population, maintaining clean resources and food qualities, and protecting climate and environment. Food sustainability is mostly a cooperative effort resulting in technology development supported by both governments and enterprises. Multiple attempts have been promoted in tackling challenges and enhancing drivers in food production. Biosensors and biosensing technologies with their applications, are being widely applied to tackling top challenges in food production and its sustainability. Consequently, a growing demand in biosensing technologies exists in food sustainability. Microfluidics represents a technological system integrating multiple technologies. Nanomaterials, with its technology in biosensing, is thought to be the most promising tool in dealing with health, energy, and environmental issues closely related to world populations. The demand of point of care (POC) technologies in this area focus on rapid, simple, accurate, portable, and low-cost analytical instruments. This review provides current viewpoints from the literature on biosensing in food production, food processing, safety and security, food packaging and supply chain, food waste processing, food quality assurance, and food engineering. The current understanding of progress, solution, and future challenges, as well as the commercialization of biosensors are summarized.

Optoelectronic fowl adenovirus detection based on local electric field enhancement on graphene quantum dots and gold nanobundle hybrid.

An optoelectronic sensor is a rapid diagnostic tool that allows for an accurate, reliable, field-portable, low-cost device for practical applications. In this study, template-free In situ gold nanobundles (Au NBs) were fabricated on an electrode for optoelectronic sensing of fowl adenoviruses (FAdVs). Au NB film was fabricated on carbon electrodes working area using L(+) ascorbic acid, gold chroloauric acid and poly-l-lysine (PLL) through modified layer-by-layer (LbL) method. A scanning electron microscopic (SEM) image of the Au NBs revealed a NB-shaped Au structure with many kinks on its surface, which allow local electric field enhancement through light-matter interaction with graphene quantum dots (GQDs). Here, GQDs were synthesized through an autoclave-assisted method. Characterization experiments revealed blue-emissive, well-dispersed GQDs that were 2-3nm in size with the fluorescence emission peak of GQDs located at 405nm. Both Au NBs and GQDs were conjugated with target FAdVs specific antibodies that bring them close to each other with the addition of target FAdVs through antibody-antigen interaction. At close proximity, light-matter interaction between Au NBs and QDs produces a local electric signal enhancement under Ultraviolet-visible (UV-visible) light irradiation that allows the detection of very low concentrations of target virus even in complex biological media. A proposed optoelectronic sensor showed a linear relationship between the target FAdVs and the electric signal up to 10 Plaque forming unit (PFU)/mL with a limit of detection (LOD) of 8.75 PFU/mL. The proposed sensing strategy was 100 times more sensitive than conventional ELISA method.

Microfluidic platform integrated with graphene-gold nano-composite aptasensor for one-step detection of norovirus.

Noroviruses are a foremost cause of gastroenteritis outbreaks throughout the world. On-site sample processing and detection of the viral clinical samples has always been a problem. This study reports an all-polydimethylsiloxane microfluidic chip integrated with screen-printed carbon electrode for the electrochemical detection of norovirus. The microfluidic chip contained packed silica microbeads zones to filter and enrich the norovirus infected clinical sample. Selective detection of norovirus was accomplished by functionalizing the graphene-gold nanoparticles composite modified carbon electrode with the viral capsid-specific aptamer. Norovirus specific aptamer was tagged with a ferrocene molecule, which acts a redox probe. The interaction of aptamer and norovirus resulted in a decrease in the electrochemical signal from ferrocene. The microfluidic chip and functionalized electrodes were characterized using several microscopic and electrochemical techniques. The optimized microfluidic aptasensor was employed to detect a range of norovirus concentration. Using differential pulse voltammetric analysis, a detection limit of 100 pM with a detection range from 100 pM to 3.5nM for norovirus was obtained. The application of aptasensor was also assessed by detecting norovirus in spiked blood samples. The aptasensor could easily discriminate between the target norovirus and other interfering molecules. The developed microfluidic aptasensor has the potential to be used for point-of-care one-step detection of norovirus in clinical samples.

Microscale loop-mediated isothermal amplification of viral DNA with real-time monitoring on solution-gated graphene FET microchip.

Rapid and reliable molecular analysis of DNA for disease diagnosis is highly sought-after. FET-based sensors fulfill the demands of future point-of-care devices due to its sensitive charge sensing and possibility of integration with electronic instruments. However, most of the FETs are unstable in aqueous conditions, less sensitive and requires conventional Ag/AgCl electrode for gating. In this work, we propose a solution-gated graphene FET (SG-FET) for real-time monitoring of microscale loop-mediated isothermal amplification of DNA. The SG-FET was fabricated effortlessly with graphene as an active layer, on-chip co-planar electrodes, and polydimethylsiloxane-based microfluidic reservoir. A linear response of about 0.23V/pH was seen when the buffers from pH 5-9 were analyzed on the SG-FET. To evaluate the performance of SG-FET, we monitored the amplification of Lambda phage gene as a proof-of-concept. During amplification, protons are released, which gradually alters the Dirac point voltage (VDirac) of SG-FET. The resulting device was highly sensitive with a femto-level limit of detection. The SG-FET could easily produce a positive signal within 16.5min of amplification. An amplification of 10ng/µl DNA for 1h produced a ∆VDirac of 0.27V. The sensor was tested within a range of 2×102 copies/µl (10 fg/µl) to 2×108 copies/µl (10ng/µl) of target DNA. Development of this sensing technology could significantly lower the time, cost, and complications of DNA detection.

Recent Advances in Biosensor Development for Foodborne Virus Detection.

Outbreaks of foodborne diseases related to fresh produce have been increasing in North America and Europe. Viral foodborne pathogens are poorly understood, suffering from insufficient awareness and surveillance due to the limits on knowledge, availability, and costs of related technologies and devices. Current foodborne viruses are emphasized and newly emerging foodborne viruses are beginning to attract interest. To face current challenges regarding foodborne pathogens, a point-of-care (POC) concept has been introduced to food testing technology and device. POC device development involves technologies such as microfluidics, nanomaterials, biosensors and other advanced techniques. These advanced technologies, together with the challenges in developing foodborne virus detection assays and devices, are described and analysed in this critical review. Advanced technologies provide a path forward for foodborne virus detection, but more research and development will be needed to provide the level of manufacturing capacity required.

Highly selective organic transistor biosensor with inkjet printed graphene oxide support system.

Most of the reported field effect transistors (FETs) fall short of a general method to uniquely specify and detect a target analyte. For this reason, we propose a pentacene-based FET with a graphene oxide support system (GOSS), composed of functionalized graphene oxide (GO) ink. The GOSS with a specific moiety group to capture the biomaterial of interest was inkjet printed on the pentacene FET. It provided modular receptor sites on the surface of pentacene, without alteration of the device. To evaluate the performance of a GOSS-pentacene FET biosensor, we detected the artificial DNA and circulating tumor cells as a proof-of-concept. The mobility of the FET dramatically changed upon capturing the target biomolecule on the GOSS. The FET exhibited high selectivity with 0.1 pmoles of the target DNA and a few cancer cells per detection volume. This study suggests a valuable sensor for medical diagnosis that can be mass produced effortlessly at low-cost.

Rapid Detection of Protein Kinase on Capacitive Sensing Platforms.

In this study, we developed a capacitive sensor for the one-step and label-free detection of protein kinase A (PKA) enzyme. Metal-insulator-semiconductor (MIS) and electrolyte-insulator-semiconductor (EIS) are a simple electronic transducer, which allows efficient detection of the target analyte. For this reason, we performed a comparative sensing of PKA on the MIS and EIS capacitive sensor. The PKA-specific aptamer was used for the one-step detection. For the immobilization of thiolated aptamer, the MIS sensor contained a thin gold layer, whereas the EIS sensor had a self-aligned monolayer of gold nanoparticles. The interaction of aptamer and PKA changed the charge and density of the sensor surface. The quantitative detection of PKA was performed by analyzing the capacitance-voltage curve after the aptamer-PKA interaction. The MIS and EIS sensor showed a detection limit of 5 U/mL and 1 U/mL, respectively, for the detection of PKA. This study suggests valuable sensing platforms for the rapid and sensitive biochemical diagnosis.

Rhodium Complex and Enzyme Couple Mediated Electrochemical Detection of Adenosine.

Adenosine is one of the nucleoside which plays an important role in signal transduction and neuromodulation. This work proposes a simple electrochemical assay, comprising two enzymes and rhodium complex based electron transfer mediator, for the detection of adenosine. Sequential reaction of adenosine deaminase and L-glutamic dehydrogenase and the supporting cycle between ß-NADH and mediator enable quantitative analysis of adenosine. Role of electron transfer mediator is the conveyance of proton from electrode to ß-NAD(+) for regeneration of ß-NADH. The electrochemical characteristics of electron transfer mediator were also studied. Real-time adenosine detection was carried out using this multiple enzyme based chronoamperometric assay. The analysis results show a low limit of detection (140 µM) and good correspondence between current signal and the adenosine concentration (R (2) = 0.997).

Analytical detection of biological thiols in a microchip capillary channel.

Sulfur-containing amino acids, such as cysteine and homocysteine play crucial roles in biological systems for the diagnosis of medical states. In this regard, this paper deals with separation, aliquot and detection of amino thiols on a microchip capillary electrophoresis with electrochemical detection in an inverted double Y-shaped microchannel. Unlike the conventional capillary electrophoresis, the modified microchannel design helps in storing the separated thiols in different reservoirs for further analysis, if required; and also eliminates the need of electrodes regeneration. The device was fabricated using conventional photolithographic technique which consisted of gold microelectrodes on a soda lime glass wafer and microchannels in PDMS mold. Multiple detections were performed using in-house fabricated dual potentiostat. Based on amperometric detection, cysteine and homocysteine were analyzed in 105 s and 120 s, respectively after diverting in branched channels. Repeated experiments proved the good reproducibility of the device. The device produced a linear response for both cysteine and homocysteine in electrochemical analysis. To prove the practicality of device, we also analyzed cysteine and homocysteine in real blood samples without any pre-treatment. Upon calculation, the device showed a very low limit of detection of 0.05 µM. The modified microchip design shall find a broad range of analytical applications involving assays of thiols and other biological compounds.