Nanozyme-assisted molecularly imprinted polymer-based indirect competitive ELISA for the detection of marine biotoxin.

Saxitoxin (STX), which is produced by certain dinoflagellate species, is a type of paralytic shellfish poisoning toxin that poses a serious threat to human health and the environment. Therefore, developing a technology for the convenient and cost-effective detection of STX is imperative. In this study, we developed an affinity peptide-imprinted polymer-based indirect competitive ELISA (ic-ELISA) without using enzyme-toxin conjugates. AuNP/Co3O4@Mg/Al cLDH was synthesized by calcining AuNP/ZIF-67@Mg/Al LDH, which was obtained by combining AuNPs, ZIF-67, and flower-like Mg/Al LDH. This synthesized nanozyme exhibited high catalytic activity (Km = 0.24 mM for TMB and 132.5 mM for H2O2). The affinity peptide-imprinted polymer (MIP) was imprinted with an STX-specific template peptide (STX MIP) on a multi-well microplate and then reacted with an STX-specific signal peptide (STX SP). The interaction between the STX SP and MIP was detected using a streptavidin-coated nanozyme (SA-AuNP/Co3O4@Mg/Al cLDH). The developed MIP-based ic-ELISA exhibited excellent selectivity and sensitivity, with a limit of detection of 3.17 ng/mL (equivalent: 0.317 µg/g). Furthermore, the system was validated using a commercial ELISA kit and mussel tissue samples, and it demonstrated a high STX recovery with a low coefficient of variation. These results imply that the developed ic-ELISA can be used to detect STX in real samples.

Green Synthetic Approach for the Preparation of Blue Emitting Gold Nanoclusters: A Simple Analytical Method for Detection of Hexaconazole Fungicide.

Blue emissive Argyreia nervosa-capped gold nanoclusters (A. nervosa-AuNCs) were synthesized via a simple environment-friendly method. The developed probe exhibits rapid response towards the target analyte (hexaconazole fungicide). Several characterizations, including FT-IR, UV-visible, fluorescence, HR-TEM, XPS, and fluorescence lifetime, were studied to confirm the formation of A. nervosa-AuNCs. The A. nervosa-AuNCs displayed emission and excitation peaks at 470 and 390 nm, respectively. Furthermore, the quantum yield (QY) of A. nervosa-AuNCs was 21.25%. The as-synthesized A. nervosa-AuNCs showed a good linear response with hexaconazole in the concentration range of 0.025-180 µM, with a detection limit (LOD) of 21.94 nM, indicating A. nervosa-AuNCs could be used as a sensitive and selective probe for detecting hexaconazole through a fluorescence "turn-off" mechanism. The A. nervosa-AuNCs were successfully used to detect hexaconazole in real samples. Moreover, A. nervosa-AuNCs were used as a bio-imaging probe for visualization of Saccharomyces cerevisiae cells.

Fluorescence 'turn-off-on' assays for neomycin sulphate and K+ ions with orange-red fluorescent molybdenum nanoclusters.

Fluorescent metal nanoclusters (MNCs) have found extensive application in recognizing molecular species. Here, orange-red fluorescent Arg-A. paniculata-MoNCs were synthesized using Andrographis paniculata leaf extract, arginine as a ligand, and MoCl5 as a metal precursor. The Arg-A. paniculata-MoNCs complex exhibited a quantum yield (QY) of 16.91% and excitation/emission wavelengths of 400/665 nm. The synthesized Arg-A. paniculata-MoNCs successfully acted as a probe for assaying neomycin sulphate (NS) via fluorescence turn-off and K+ ions via fluorescence turn-on mechanisms, respectively. Moreover, the developed probe was effectively used to develop a cellulose paper strip-based sensor for detection of NS and K+ ions. Arg-A. paniculata-MoNCs demonstrated great potential for sensing NS and K+ ions, with concentration ranges of 0.1-80 and 0.25-110 µM for NS and K+ ions, respectively. The as-synthesized Arg-A. paniculata-MoNCs efficiently detected NS and K+ ions in food and biofluid samples, respectively.

Green Emissive Molybdenum Nanoclusters for Selective and Sensitive Detection of Hydroxyl Radical in Water Samples.

In this work, Cassia tora (C. tora) have been used as a template to synthesize green fluorescent C. tora molybdenum nanoclusters (C. tora-MoNCs) through a green chemistry approach. These C. tora-MoNCs showed a quantum yield (QY) of 7.72% and exhibited a significant emission peak at 498 nm when excited at 380 nm. The as-prepared C. tora-MoNCs had an average size of 3.48 ± 0.80 nm and showed different surface functionality. The as-synthesized C. tora-MoNCs were successfully identified the hydroxyl radical (•OH) via a fluorescence quenching mechanism. Also, fluorescence lifetime and Stern-Volmer proved that after the addition of •OH radicals it was quenched the fluorescence intensity via a static quenching mechanism. The limit of detection is 9.13 nM, and this approach was successfully utilized for sensing •OH radicals in water samples with a good recovery rate.

Serodiagnosis of multiple cancers using an extracellular protein kinase A autoantibody-based lateral flow platform.

Extracellular protein kinase A autoantibody (ECPKA-AutoAb) has been suggested as a universal cancer biomarker due to its higher amounts in serum of several types of cancer patients than that of normal individuals. Herein, we first developed a lateral flow immunoassay (LFIA) tool, using a sandwich format, toward ECPKA-AutoAb in human serum. For this format, 3G2 as a capture antibody was identified using hybridoma technique and a series of screenings where it showed superior capacity to recognize Enzo PKA catalytic subunit alpha (Cα), compared to other PKA antibodies and antigens. Using these components, we performed sandwich ELISA toward a mimic and real sample of ECPKA-AutoAb. As per the results, limit of detection (LOD) was found to be 135 ng/mL and ECPKA-AutoAb levels were higher in various cancer patients than in normal individuals like previous studies. Based on these results, we applied this sandwich format into LFIA tool and found that the LOD of the fabricated LFIA tool showed about 3.8 ng/mL using spiked PKA-Ab, which is significantly improved compared to the LOD of sandwich ELISA. Also, the developed LFIA tool demonstrated a remarkable ability to detect significant differences in ECPKA-AutoAb levels between normal and cancer patients within 15 min, showing a potential for point-of-care (PoC) detection. One interesting point is that our LFIA strip contains an additional conjugation pad II, named because of its position behind the conjugation pad, in which PKA Cα is dried, enabling a sandwich format.

Development of Simple Fluorescence Analytical Strategy for the Detection of Triazophos Using Greenish-Yellow Emissive Carbon Dots Derived from Curcuma longa.

This study describes a new method for synthesizing water-soluble carbon dots (CDs) using "Curcuma longa" (green source) named CL-CDs via a single-step hydrothermal process. The as-synthesized CL-CDs exhibited greenish-yellow fluorescence at 548 nm upon excitation at 440 nm. It shows good water stability and exhibits a quantum yield of 19.4%. The developed probe is utilized for sensing triazophos (TZP) pesticide via a dynamic quenching mechanism, exhibiting favorable linearity ranging from 0.5-500 µM with a limit of detection of 0.0042 µM. The as-prepared CL-CDs probe was sensitive and selective towards TZP. Lastly, the successful application of the CL-CDs-based fluorescent probe in water and rice samples highlights its potential as a reliable and efficient method for the detection of TZP in various real sample matrices. Eventually, bioimaging and biocompatibility aspects of CL-CDs have been assessed on Saccharomyces cerevisiae (yeast) cell and lung cancer (A549) cell lines, respectively.

Development of Copper Nanoclusters-Based Turn-Off Nanosensor for Fluorescence Detection of Two Pyrethroid Pesticides (Cypermethrin and Lambda-Cyhalothrin).

Fluorescent copper nanoclusters (Cu NCs) were synthesized by using Withania somnifera (W. somnifera) plant extract as a biotemplate. Aqueous dispersion of W. somnifera-Cu NCs displays intense emission peak at 458 nm upon excitation at 350 nm. This fluorescence emission was utilized for the detection of two pyrethroid pesticides (cypermethrin and lambda-cyhalothrin) via "turn-off" mechanism. Upon the addition of two pyrethiod pesticides independently, the fluorescence emission of W. somnifera-Cu NCs was gradually decreased with increasing concentrations of both pesticides. It was noticed that the decrease in emission intensity at 458 nm was linearly dependent on the logarithm of both pesticides concentrations in the ranges of 0.01-100 µM and of 0.05-100 µM for cypermethrin and lambda-cyhalothrin, respectively. Consequently, the limits of detection were found to be 27.06 and 23.28 nM for cypermethrin and lambda-cyhalothrin, respectively. The as-fabricated W. somnifera-Cu NCs acted as a facile sensor for the analyses of cypermethrin and lambda-cyhalothrin in vegetables (tomato and bottle gourd), which demonstrates that it could be used as portable sensing platform for assaying of two pyrethroid pesticides in food samples.

Synthesis of Greenish-Yellow Fluorescent Copper Nanocluster for the Selective and Sensitive Detection of Fipronil Pesticide in Vegetables and Grain Samples.

In this paper, a new synthetic route is introduced for the synthesis of high-luminescent greenish-yellow fluorescent copper nanoclusters (PVP@A. senna-Cu NCs) using Avaram senna (A. senna) and polyvinylpyrrolidone (PVP) as templates. A. senna plant extract mainly contains variety of phytochemicals including glycosides, sugars, saponins, phenols, and terpenoids that show good pharmacological activities such as anti-inflammatory, antioxidant, and antidiabetic. PVP is a stable and biocompatible polymer that is used as a stabilizing agent for the synthesis of PVP@A. senna-Cu NCs. The size, surface functionality, and element composition of the fabricated Cu NCs were confirmed by various analytical techniques. The as-prepared greenish-yellow fluorescent Cu NCs exhibit significant selectivity towards fipronil, thereby favoring to assay fipronil pesticide with good linearity in the range of 3.0-30 µM with a detection limit of 65.19 nM. More importantly, PVP@A. senna-Cu NCs are successfully applied to assay fipronil in vegetable and grain samples.

Machine Learning-Based Approach to Developing Potent EGFR Inhibitors for Breast Cancer-Design, Synthesis, and In Vitro Evaluation.

The epidermal growth factor receptor (EGFR) is vital for regulating cellular functions, including cell division, migration, survival, apoptosis, angiogenesis, and cancer. EGFR overexpression is an ideal target for anticancer drug development as it is absent from normal tissues, marking it as tumor-specific. Unfortunately, the development of medication resistance limits the therapeutic efficacy of the currently approved EGFR inhibitors, indicating the need for further development. Herein, a machine learning-based application that predicts the bioactivity of novel EGFR inhibitors is presented. Clustering of the EGFR small-molecule inhibitor (∼9000 compounds) library showed that N-substituted quinazolin-4-amine-based compounds made up the largest cluster of EGFR inhibitors (∼2500 compounds). Taking advantage of this finding, rational drug design was used to design a novel series of 4-anilinoquinazoline-based EGFR inhibitors, which were first tested by the developed artificial intelligence application, and only the compounds which were predicted to be active were then chosen to be synthesized. This led to the synthesis of 18 novel compounds, which were subsequently evaluated for cytotoxicity and EGFR inhibitory activity. Among the tested compounds, compound 9 demonstrated the most potent antiproliferative activity, with 2.50 and 1.96 µM activity over MCF-7 and MDA-MB-231 cancer cell lines, respectively. Moreover, compound 9 displayed an EGFR inhibitory activity of 2.53 nM and promising apoptotic results, marking it a potential candidate for breast cancer therapy.

Synthesis of copper nanoclusters from Bacopa monnieri leaves for fluorescence sensing of dichlorvos.

In this work, a facile one-step green synthesis was developed for the fabrication of blue fluorescent copper nanocluster (Brahmi-CuNCs) from the extract of Bacopa monnieri (common name is Brahmi) via a microwave method. The as-prepared Brahmi-CuNCs emitted blue fluorescence at 452 nm when excited at 352 nm and showed a quantum yield of 31.32%. Brahmi-derived blue fluorescent CuNCs acted as a probe for fluorescence sensing of dichlorvos. Upon the addition of dichlorvos, the blue emission for Brahmi-CuNCs was gradually turned off, favouring establishment of a calibration graph in the range 0.5-100 µM with a detection limit of 0.23 µM. The as-synthesized Brahmi-CuNCs exhibited marked sensitivity and selectivity towards dichlorvos, favourable for assaying dichlorvos in various samples (cabbage, apple juice, and rice).

Highly sensitive electrochemical peptide-based biosensor for marine biotoxin detection using a bimetallic platinum and ruthenium nanoparticle-tethered metal-organic framework modified electrode.

Saxitoxin (STX) is a highly toxic small-molecule cyanotoxin that is water-soluble, stable in acidic media, and thermostable. STX is hazardous to human health and the environment in ocean, thus it is an important to detect it at very low concentrations. Herein, we developed an electrochemical peptide-based biosensor for the trace detection of STX in different sample matrix utilizing differential pulse voltammetry (DPV) signal. We synthesized the nanocomposite of zeolitic imidazolate framework-67 (ZIF-67) decorated bimetallic platinum (Pt) and ruthenium (Ru) nanoparticles (Pt-Ru@C/ZIF-67) using impregnation method. The nanocomposite modified with screen-printed electrode (SPE) was subsequently used to detect STX in the range of 1-1,000 ng mL-1, with a detection limit (LOD) of 26.7 pg mL-1. The developed peptide-based biosensor is highly selective and sensitive towards STX detection, thus it represents a promising strategy for the development of novel portable bioassay for monitoring various hazardous molecules in aquatic food chains.

Fluorescence "Turn OFF-ON" detection of Fe3+ and propiconazole pesticide using blue emissive carbon dots from lemon peel.

In this study, water-soluble carbon dots (CDs) were employed as a novel fluorescence "turn OFF-ON" sensor to detect Fe3+ ions in pharmaceutical sample and propiconazole (PC) in food samples. Blue fluorescent "LPCDs" are synthesized from the lemon peel that exhibited emission at 468 nm when excited at 378 nm. The average size of the as-prepared LPCDs is 2.03 nm, displaying a quantum yield of 32 %. Fluorescence "turn OFF-ON" strategy was developed for sensing of Fe3+ ion and PC, demonstrating favorable linearity in the range of 0.5-180 µM and 0.1-40 µM with the detection limits of 0.18 µM and 0.054 µM for Fe3+ and PC, respectively. Further, LPCDs-loaded cellulose paper was used as visual reader to detect Fe3+ and PC. This approach was effectively applied to detect Fe3+ and PC in pharmaceutical and vegetable samples.

Synthesis of multicolor silver nanostructures for colorimetric sensing of metal ions (Cr3+, Hg2+ and K+) in industrial water and urine samples with different spectral characteristics.

In this work, we have synthesized four different color (yellow, orange, green, and blue (multicolor)) silver nanostructures (AgNSs) by chemical reduction method where silver nitrate, sodium borohydride and hydrogen peroxide were used as reagents. The as-synthesized multicolor AgNSs were successfully functionalized with bovine serum albumin (BSA) and applied as a colorimetric sensor for the assaying of metal cations (Cr3+, Hg2+, and K+). The addition of metal ions (Cr3+, Hg2+, and K+) into BSA functionalized AgNSs (BSA-AgNSs) causes the aggregation of BSA-AgNSs, and are accompanied by visual color changes with red or blue shift in the surface plasmon resonance (SPR) band of BSA-AgNSs. The BSA-AgNSs show different SPR characteristic for each metal ions (Cr3+, Hg2+, and K+) with exhibiting different spectral shift and color change. The yellow color BSA-AgNSs (Y-BSA-AgNSs) act as a probe for sensing Cr3+, orange color BSA-AgNSs (O-BSA-AgNSs) act as probe for Hg2+ ion assay, green color BSA-AgNSs (G-BSA-AgNSs) act as a probe for the assaying of both K+ and Hg2+, and blue color BSA-AgNSs (B-BSA-AgNSs) act as a sensor for colorimetric detection of K+ ion. The detection limits were found to be 0.26 µM for Cr3+ (Y-BSA-AgNSs), 0.14 µM for Hg2+ (O-BSA-AgNSs), 0.05 µM for K+ (G-BSA-AgNSs), 0.17 µM for Hg2+ (G-BSA-AgNSs), and 0.08 µM for K+ (B-BSA-AgNSs), respectively. Furthermore, multicolor BSA-AgNSs were also applied for assaying of Cr3+, and Hg2+ in industrial water samples and K+ in urine sample.

Highly sensitive and label-free electrochemical detection of C-reactive protein on a peptide receptor-gold nanoparticle-black phosphorous nanocomposite modified electrode.

C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein found in blood serum, and an enhanced CRP level is indicative of inflammatory conditions such as infection and cancer, among others. In this work, we developed a novel high CRP-affinity peptide-functionalized label-free electrochemical biosensor for the highly sensitive and selective detection of CRP. Throughout biopanning with random peptide libraries, high affinity peptides for CRP was successfully identified, and then a series of synthetic peptide receptor, of which C-terminus was incorporated to gold binding peptide (GBP) as an anchoring motif was covalently immobilized onto gold nanoparticle (AuNPs) tethered polydopamine (PDA)‒black phosphorus (BP) (AuNPs@BP@PDA) nanocomposite electrode. Interaction between the CRP-binding peptide and CRP was confirmed via enzyme-linked immunosorbent assay along with various physicochemical and electrochemical analyses. Under the optimized experimental conditions, the proposed peptide-based biosensor detects CRP in the range of 0-0.036 µg/mL with a detection limit (LOD) of 0.7 ng/mL. The developed sensor effectively detects CRP in the real samples of serum and plasma of Crohn's disease patients. Thus, the fabricated peptide-based biosensor has potential applications in clinical diagnosis and medical applications.

Antibody-free and selective detection of okadaic acid using an affinity peptide-based indirect assay.

Okadaic acid (OA) is a type of marine biotoxin produced by some species of dinoflagellates in marine environments. Consumption of shellfish contaminated with OA can cause diarrhetic shellfish poisoning (DSP) in humans with symptoms that typically include abdominal pain, diarrhea and vomiting. In this study, we developed an affinity peptide-based direct competition enzyme-linked immunosorbent assay (dc-ELISA) for the detection of OA in real samples. The OA-specific peptide was successfully identified via M13 biopanning and a series of peptides were chemically synthesized and characterized their recognition activities. The dc-ELISA system showed good sensitivity and selectivity with a half-maximal inhibitory concentration (IC50) of 148.7 ng/mL and a limit of detection (LOD) of 5.41 ng/mL (equivalent, 21.52 ng/g). Moreover, the effectiveness of the developed dc-ELISA was validated using OA-spiked shellfish samples, and the developed dc-ELISA showed a high recovery rate. These results suggest that the affinity peptide-based dc-ELISA can be a promising tool for detecting OA in shellfish samples.

Development of new TAK-285 derivatives as potent EGFR/HER2 inhibitors possessing antiproliferative effects against 22RV1 and PC3 prostate carcinoma cell lines.

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) protein tyrosine kinases co-expressed in various cancers such as ovarian, breast, colon, and prostate subtypes. Herein, new TAK-285 derivatives (9a-h) were synthesised, characterised, and biologically evaluated as dual EGFR/HER2 inhibitors. Compound 9f exhibited IC50 values of 2.3 nM over EGFR and 234 nM over HER2, which is 38-fold of staurosporine and 10-fold of TAK-285 over EGFR. Compound 9f also showed high selectivity profile when tested over a small kinase panel. Compounds 9a-h showed IC50 values in the range of 1.0-7.3 nM and 0.8-2.8 nM against PC3 and 22RV1 prostate carcinoma cell lines, respectively. Cell cycle analysis, apoptotic induction, molecular docking, dynamics, and MM-GBSA studies confirmed the plausible mechanism(s) of compound 9f as a potent EGFR/HER2 dual inhibitor with an effective antiproliferative action against prostate carcinoma.

Transition metal complex-incorporated polyaniline as a platform for an enzymatic uric acid electrochemical sensor.

Uric acid (UA) is the primary waste product from purine metabolism in humans. Excessive UA levels in the body will accumulate in joints and form crystals that cause a wide range of health problems. An enzymatic electrochemical biosensor for UA based on the transition metal complex-incorporated polyaniline PANI-RC functionalized with both urate oxidase (UOx) as a specific bioreceptor and horseradish peroxidase (HRP) as a signal enhancer was developed. The transition metal complex being used herein is the commonly used redox couple (RC) in electrochemical biosensors, [Fe(CN)6]3-/4-, which plays the pivotal role of electron acceptors. This PANI-RC platform then becomes a conducive environment not only for enzyme immobilization but also for signal transfer improvement. The synergistic combination of HRP near UOx and RC anchored on the backbone of PANI helps in electron transfer from the enzymatic reaction to the current collector. The resulting PANI-RC-based UA sensor demonstrates high sensitivity with a detection limit of 11.4 µM, wide linear range, good stability, and excellent selectivity even in the presence of the most problematic interference in UA assays (e.g., ascorbic acid and urea). The recovery tests using artificial biofluid-spiked UA samples also showed promising results for practical usage of the PANI-RC-based UA sensor.

Sensitive Detection of BVDV Using Gold Nanoparticle-Modified Few-Layer Black Phosphorus with Affinity Peptide-Based Electrochemical Sensor.

The lethality of the bovine viral diarrhea virus (BVDV) in cattle involves inapparent infection and various, typically subclinical, syndromes. Cattle of all ages are vulnerable to infection with the virus. It also causes considerable economic losses, primarily due to reduced reproductive performance. In the absence of treatment that can completely cure infected animals, detection of BVDV relies on highly sensitive and selective diagnosis methods. In this study, an electrochemical detection system was developed as a useful and sensitive system for the detection of BVDV to suggest the direction of diagnostic technology through the development of conductive nanoparticle synthesis. As a countermeasure, a more sensitive and rapid BVDV detection system was developed using the synthesis of electroconductive nanomaterials black phosphorus (BP) and gold nanoparticle (AuNP). To increase the conductivity effect, AuNP was synthesized on the BP surface, and the stability of BP was improved by using dopamine self-polymerization. Moreover, its characterizations, electrical conductivity, selectivity, and sensitivity toward BVDV also have been investigated. The BP@AuNP-peptide-based BVDV electrochemical sensor exhibited a low detection limit of 0.59 copies mL-1 with high selectivity and long-term stability (retaining 95% of its initial performance over 30 days).

Highly sensitive and label-free detection of influenza H5N1 viral proteins using affinity peptide and porous BSA/MXene nanocomposite electrode.

Influenza viruses are known to cause pandemic flu through inter-human and animal-to-human transmissions. Neuraminidase (NA), which is a surface glycoprotein of both influenza A and B viruses, is a minor immunogenic determinant; however, it has been proposed as an ideal candidate for a real testing. We successfully identified an affinity peptide which is specific to the influenza H5N1 virus NA via phage display technique and observed initially its binding affinities using enzyme-linked immunosorbent assay (ELISA). In addition, four synthetic peptides were chemically synthesized to develop an affinity peptide-based electrochemical biosensing system. Among all peptides tested, INA BP2 was selected as a potential candidate and subjected to square-wave voltammetry (SWV) for evaluating their detection performance. To enhance analytical performance, a three-dimensional porous bovine serum albumin (BSA)-MXene (BSA/MXene) matrix was applied. The surface morphology of the BSA/MXene film-deposited electrode was analyzed using X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Using SWV measurement, the BSA/MXene nanocomposite-based peptide sensor exhibited significant the dissociation constant (Kd = 9.34 ± 1.20 nM) and the limit of detection (LOD, 0.098 nM), resulting in good reproducibility, stability and recovery, even in the presence with spiked human plasma. These results demonstrate an alternative way of new bioanalytical sensing platform for developing more desirable sensitivity in other virus detection.