A Quadruplex Ultrasensitive Immunoassay for Simultaneous Assessment of Human Reproductive Hormone Proteins in Multiple Biofluid Samples.

Reproductive hormones play vital roles in reproductive health and can be used to assess a woman's ovarian function and diagnose diseases associated with reproductive endocrine disorders. As these hormones are important biomarkers for reproductive health monitoring and diagnosis, a rapid, high-throughput, and low-invasive detection and simultaneous assessment of the levels of multiple reproductive hormones has important clinical applications. In this work, a quadruplex ultrasensitive immunoassay was developed for simultaneous assessment of 4 human reproductive hormone proteins (follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and anti-Müllerian hormone (AMH)) in a variety of human biofluid samples. This assay takes advantage of single-molecule imaging of microwell arrays and capture antibody beads as a reaction interface to construct multiplex bead array immunoassays. The analyte-bound beads can easily be parsed to individual wells and detected via fluorophores, emitting distinct wavelengths associated to the beads. As a result, this proposed quadruplex immunoassay exhibits four good 4-parameter logistic calibration curves ranging from 2.7 to 2000, 1.6 to 1200, 1.8 to 1300, and 0.3 to 220 pg/mL with limits of detection of 0.32, 0.28, 0.14, and 0.02 pg/mL for FSH, LH, PRL, and AMH, respectively. Furthermore, the developed quadruplex immunoassay was used to test clinical venous serum samples where it showed remarkable consistency with clinical test results in methodological comparison and the diagnosis of polycystic ovary syndrome. In addition, we successfully applied the ultrasensitive capability of this assay to the simultaneous testing and evaluation of four proteins in fingertip blood as well as urine samples, in which the urinary AMH level (1.42-156 pg/mL) was measured and assessed quantitatively for the first time.

Salts-assistant synthesis of g-C3N4/Prussian-blue analogue/nickel foam with hierarchical structures as binder-free electrodes for supercapacitors.

The exploitation of high-performance electrode materials is significant to develop supercapacitors with satisfied energy and power output properties. In this study, a g-C3N4/Prussian-blue analogue (PBA)/Nickel foam (NF) with hierarchical micro/nano structures was developed by a simple salts-directed self-assembly approach. In this synthetic strategy, NF acted as both 3D macroporous conductive substrate and Ni source for PBA formation. Moreover, the incidental salt in molten salt-synthesized g-C3N4 nanosheets could regulate the combination mode between g-C3N4 and PBA to generate interactive networks of g-C3N4 nanosheets-covered PBA nano-protuberances on NF surfaces, which further expended the electrode/electrolyte interfaces. Based on the merits from this unique hierarchical structure and the synergy effect of PBA and g-C3N4, the optimized g-C3N4/PBA/NF electrode exhibited a maximum areal capacitance of 3366 mF cm-2 at current of 2 mA cm-2, as well as 2118 mF cm-2 even under large current of 20 mA cm-2. The solid-state asymmetric supercapacitor using g-C3N4/PBA/NF electrode possessed an extended working potential window of 1.8 V, prominent energy density of 0.195 mWh cm-2 and power density of 27.06 mW cm-2. Compared to the device with pure NiFe-PBA electrode, a better cyclic stability with capacitance retention rate of 80% after 5000 cycles was also achieved due to the protective effect of g-C3N4 shells on the etching of PBA nano-protuberances in electrolyte. This work not only builds a promising electrode material for supercapacitors, but also provide an effective way to apply molten salt-synthesized g-C3N4 nanosheet without purification.

Highly sensitive and ratiometric detection of nitrite in food based on upconversion-carbon dots nanosensor.

Nitrite (NO2-) is extensively found in the daily dietary environment. However, consuming too much NO2- can pose serious health risks. Thus, we designed a NO2--activated ratiometric upconversion luminescence (UCL) nanosensor which could realize NO2- detection via the inner filter effect (IFE) between NO2--sensitive carbon dots (CDs) and upconversion nanoparticles (UCNPs). Due to the exceptional optical properties of UCNPs and the remarkable selectivity of CDs, the UCL nanosensor exhibited a good response to NO2-. By taking advantage of NIR excitation and ratiometric detection signal, the UCL nanosensor could eliminate the autofluorescence thereby increasing the detection accuracy effectively. Additionally, the UCL nanosensor proved successful in detecting NO2- quantitatively in actual samples. The UCL nanosensor provides a simple as well as sensitive sensing strategy for NO2- detection and analysis, which is anticipated to extend the utilization of upconversion detection in food safety.

Runx1/miR-26a/Jagged1 signaling axis controls osteoclastogenesis and alleviates orthodontically induced inflammatory root resorption.

BACKGROUND: MicroRNAs (miRNAs) are involved in the regulation of osteoclast biology and several pathogenic progression. This study aimed to identify the role of miR-26a in osteoclastogenesis and orthodontically induced inflammatory root resorption(OIIRR). METHODS: Rat orthodontic tooth movement (OTM) model was established by ligating a closed coil spring between maxillary first molar and incisor, and 50 g orthodontic force was applied to move upper first molar to middle for 7 days. Human periodontal ligament (hPDL) cells were isolated from periodontium of healthy donors, and then subjected to compression force (CF) for 24 h to mimic an in vitro OTM model. The levels of associated factors in vivo and in vitro were measured subsequently. RESULT: The distance of tooth movement was increased and root resorption pits were occurred in rat OTM model. The expression of miR-26a was decreased in vivo and vitro experiments. CF treatment enhanced the secretion of inflammatory factors receptor activator of nuclear factor-kappa B ligand (RANKL) and IL-6, osteoclast marker levels, and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, while miR-26a overexpression reversed these results. Furthermore, miR-26a overexpression inhibited the osteoclastogenesis and rescued the root resorption in OTM rats through inhibition of Jagged1. Additionally, Runx1 could bind to miR-26a promoter and promote its expression, thereby suppressing the osteoclastogenesis. CONCLUSION: We concluded that Runx1/miR-26a/Jagged1 signaling axis restrained osteoclastogenesis and alleviated OIIRR.

Simultaneous detection of the spike and nucleocapsid proteins from SARS-CoV-2 based on ultrasensitive single molecule assays.

Nucleic acid detection technology based on polymerase chain reaction (PCR) and antibody detection based on immunochromatography still have many problems such as false negatives for the diagnosis of coronavirus disease 2019 (COVID-19). Therefore, it is of great importance to develop new techniques to improve the diagnostic accuracy of COVID-19. We herein developed an ultrasensitive, rapid, and duplex digital enzyme-linked immunosorbent assay (dELISA) for simultaneous detection of spike (S-RBD) and nucleocapsid (N) proteins of SARS-CoV-2 based on a single molecule array. This assay effectively combines magnetic bead encoding technology and the ultrasensitive detection capability of a single molecule array. The detection strategies of S-RBD protein and N-protein exhibited wide response ranges of 0.34-1065 pg/mL and 0.183-338 pg/mL with detection limits of 20.6 fg/mL and 69.8 fg/mL, respectively. It is a highly specific method for the simultaneous detection of S-RBD protein and N-protein and has minimal interference from other blood proteins. Moreover, the spike assay showed a satisfactory and reproducible recovery rate for the detection of S-RBD protein and N-protein in serum samples. Overall, this work provides a highly sensitive method for the simultaneous detection of S-RBD protein and N-protein, which shows ultrasensitivity and high signal-to-noise ratio and contributes to improve the diagnosis accuracy of COVID-19.

Field-effect transistor bioassay for ultrasensitive detection of folate receptor 1 by ligand-protein interaction.

A miniaturized and integrated bioassay was developed based on molybdenum disulfide (MoS2) field-effect transistor (FET) functionalized with bovine serum albumin-folic acid (BSA-FA) for monitoring FOLR1. We performed the electrical test of FOLR1 within the range 100 fg/mL to 10 ng/mL, and the limit of detection was 0.057 pg/mL. The ultrahigh sensitivity of the bioassay was realized by ligand-protein interaction between FA and FOLR1, with a ligand-protein binding ratio of 3:1. The formation of FA-FOLR1 was confirmed with ELISA. The binding affinity dissociation constant KD was 12 ± 6 pg/mL. This device can work well for FOLR1 detection in human serum, which presents its promising application in point-of-care diagnosis. This study supports the future applications of such ligand-protein-based bioassays in the clinical practices. Graphical abstract MoS2-based FET device for detecting folate receptor 1 (FOLR1) was fabricated. The molecular folic acid as a probe can specifically bound to FOLR1 with a high affinity.

Association of serum neurofilament light and disease severity in patients with spinocerebellar ataxia type 3.

OBJECTIVE: To investigate serum neurofilament light protein (sNfL) levels in patients with spinocerebellar ataxia type 3 (SCA3) and to determine whether they are associated with disease severity. METHODS: This cross-sectional study enrolled 185 healthy controls and 235 ATXN3 mutation carriers (17 asymptomatic stage, 20 preclinical stage, and 198 ataxic stage). We measured sNfL levels with the single molecule array (Simoa) platform. Clinical disease severity was assessed using the Scale of Assessment and Rating of Ataxia (SARA) and the Inventory of Nonataxia Signs (INAS). In a subgroup of 50 ataxic stage patients, we further evaluated the gray matter volume and the integrity of white matter fibers by MRI. RESULTS: sNfL concentrations were elevated in asymptomatic, preclinical, and ataxic ATXN3 mutation carriers compared to controls (12.18 [10.20-13.92], 21.84 [18.37-23.45], 36.06 [30.04-45.90], and 8.24 [5.92-10.84] pg/mL, median [interquartile range], respectively, p < 0.001). sNfL correlated with SARA (r = 0.406, 95% confidence interval [CI] 0.284-0.515, p < 0.0001) and INAS (r = 0.375, 95% CI 0.250-0.487, p < 0.0001), and remained significant after adjustment for age and CAG repeats. In addition, we observed negative correlations of the sNfL with gray matter volume in the left precentral gyrus and the left paracentral lobule as well as with the mean diffusivity in widespread white matter tracts. CONCLUSION: Our results demonstrate that sNfL levels are increased in SCA3 and are associated with clinical disease severity, which supports sNfL as a biomarker for disease severity in SCA3. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in patients with SCA3, sNfL elevations are associated with clinical disease severity.

A single molecule assay for ultrasensitive detection of Fn14 in human serum.

Fibroblast growth factor inducible-14 (Fn14) is a receptor protein that plays an important role in the progression of cancer and some other diseases. Here, an ultrasensitive assay was developed for the detection of Fn14 based on a digital sandwich enzyme-linked immunosorbent bead-based assay (dELISA). Beads containing a single immunocomplex are loaded into microwells (~46 fL) and produce fluorescence through enzyme-catalyzed reactions in extremely small volumes. By measuring the number of fluorescent microwells in arrays arranged on a circular Disc, the concentration of Fn14 was determined. To obtain better performance for Fn14 detection, assay conditions including reagent concentrations and measurement parameters were optimized and 44 different antibody pairs were screened. The detection range of Fn14 is 1.26 pg/mL to 3683 pg/mL with a lower detection limit of 0.32 pg/mL, which is much lower than that of conventional ELISAs. In addition, the total operation of this assay is automated and only takes approximately an hour to accomplish. Furthermore, this assay was successfully applied to the determination of spiked Fn14 in serum samples with satisfactory performance.

Highly fluorescent organic polymers for quenchometric determination of hydrogen peroxide and enzymatic determination of glucose.

Strongly fluorescent polymers (FPs) were prepared from citric acid and ethylenediamine via a hydrothermal approach. The FPs display low toxicity, water solubility, a quantum yield of 91%, good photostability and stability in the physiological pH range. Ferric ions are found to quench the fluorescence which is best measured at excitation/emission wavelengths of 350/440 nm. Because ferric ions (Fe3+) can quench the fluorescence of FPs, a fluorometric method was developed for fast detection of Fe3+ and within 1 min. FPs can also be used indirectly for the detection of hydrogen peroxide because of its fast Fenton reaction with Fe2+ to generate of Fe3+. The detection limits are 8 µM for Fe(III) and 0.6 µM for H2O2. On the basis of the glucose oxidase catalyzed of glucose and the Fenton reaction, the FPs can also be used to quantify glucose with a linear response in the 0.5-10 µM concentration range. Graphical abstract A new type of polymer with high fluorescence quantum yield was prepared. It is shown to enable the fast detection of ferric ions, hydrogen peroxide and glucose based on quenching and on the glucose oxidase and Fenton reactions.

Fluorometric determination of glucose based on a redox reaction between glucose and aminopropyltriethoxysilane and in-situ formation of blue-green emitting silicon nanodots.

A method is described for fluorometric detection of glucose. It is based on the finding that silicon nanodots (SNDs) are formed from glucose and aminopropyltriethoxysilane (APTES) under mild experimental conditions. The SNDs thus formed have an average diameter of ∼2 nm, exhibit good water dispersibility, blue fluorescence (with excitation/emission maxima at 410/475 nm), broad pH tolerance, and are photostable. The assay was applied to the quantification of glucose with high sensitivity, good specificity, and over a wide detection range (from 10 µM to 0.9 mM). It was applied to the determination of glucose in spiked serum samples and gave satisfactory results and recoveries. Graphical abstract Schematic presentation of serum glucose detection based on a redox reaction between glucose and aminopropyltriethoxysilane and in-situ formation of blue-green emitting silicon nanodots.

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

A Self-Assembly Fluorescence Sensing Platform for Glutathione Detection Based on Eco-Friendly Quantum Dots and MnO2 Nanosheets.

Glutathione (GSH), a thiol-containing tripeptide, has a pivotal role in maintaining the normally physiological environment in biology. Here, we report a novel self-assembly fluorescence resonance energy transfer (FRET) sensing platform for GSH detection by using the environment-friendly Cu:Zn-In-S/ZnS quantum dots (QDs) and MnO2 nanosheets. In this assay, fluorescence of Cu:Zn- In-S/ZnS QDs could be quenched by MnO2 nanosheets based on FRET. Upon the addition of GSH, MnO2 nanosheets were reduced into Mn2+ ions because of the rapid redox reaction, and consequently the fluorescence of QDs was recovered. A good linear relationship was obtained from 1 to 20 µM for the detection of GSH with a low detection limit of 500 nM. Meanwhile, the sensing system also exhibited high selectivity against other electrolytes and biomolecules. Moreover, the satisfactory results in the determination of GSH in serum samples indicate that the assay holds great potential for detecting GSH in biological fluids. This method shows many advantages including high sensitivity, short assay time, good simplicity as well as label-free and environmental friendly, which is expected to facilitate the GSH quantification relating to biological and biomedical studies.

Ultrasensitive determination of ascorbic acid by using cobalt oxyhydroxide nanosheets to enhance the chemiluminescence of the luminol-H2O2 system.

Ascorbic acid (AA) as an essential vitamin in the human body participates in various physiological reactions and plays a key role in many biochemical processes. Therefore, it is of vital importance to monitor and quantify AA in commercial tablets, beverages and food. In this work, a rapid and ultrasensitive chemiluminescence (CL) system for the detection of AA was developed, in which ultrathin cobalt oxyhydroxide (CoOOH) nanosheets were applied in the conventional luminol-H2O2 CL system. The results showed that ultrathin CoOOH nanosheets as a catalyzer remarkably improved the CL intensity of the CoOOH-luminol-H2O2 system, up to about 1400-fold. Under the optimized conditions, the CL inhibition efficiencies increase linearly with the concentrations of AA in the range of 1-500 pmol L-1, and the limit of detection was 39 fmol L-1. Moreover, the proposed CL system was successfully applied in the determination of AA in medicinal tablets with satisfactory results.

A facile label-free aptasensor for detecting ATP based on fluorescence enhancement of poly(thymine)-templated copper nanoparticles.

A label-free fluorescence assay has been developed for sensitive and selective detection of adenosine triphosphate (ATP) by using poly(thymine) (poly T)-templated copper nanoparticles (CuNPs) as fluorescent indicator. In our design, ATP aptamer was split into two fragments, both of which were elongated with poly T strands that can be utilized as efficient template for the formation of copper nanoparticles through the reduction of copper ions by sodium ascorbate. In the presence of ATP, the two split aptamers could be dragged to form aptamer-ATP aptamer complex, which drew the poly T strands close to each other and induced a remarkable fluorescence enhancement of poly T-templated CuNPs. Thus, an elevated fluorescence enhancement of poly T-templated CuNPs was obtained with the increase in ATP concentration. Under optimized conditions, a good linear range for ATP detection was realized from 100 nM to 100 µM with a detection limit of 10.29 nM. In addition, the application of this biosensing system in complex biological matrix was demonstrated with satisfactory results. This assay provided a simple, label-free, cost-effective, and sensitive platform for the detection of ATP.

A rapid fluorescence "switch-on" assay for glutathione detection by using carbon dots-MnO2 nanocomposites.

Glutathione (GSH) serves many cellular functions and plays crucial roles in human pathologies. Simple and sensitive sensors capable of detecting GSH would be useful tools to understand the mechanism of diseases. In this work, a rapid fluorescence "switch-on" assay was developed to detect trace amount of GSH based on carbon dots-MnO2 nanocomposites, which was fabricated through in situ synthesis of MnO2 nanosheets in carbon dots colloid solution. Due to the formation of carbon dots-MnO2 nanocomposites, fluorescence of carbon dots could be quenched efficiently by MnO2 nanosheeets through fluorescence resonance energy transfer (FRET). However, the presence of GSH would reduce MnO2 nanosheets to Mn(2+) ions and subsequently release carbon dots, which resulted in sufficient recovery of fluorescent signal. This proposed assay demonstrated highly selectivity toward GSH with a detection limit of 300nM. Moreover, this method has also shown sensitive responses to GSH in human serum samples, which indicated its great potential to be used in disease diagnosis. As no requirement of any further functionalization of these as-prepared nanomaterials, this sensing system shows remarkable advantages including very fast and simple, cost-effective as well as environmental-friendly, which suggest that this new strategy could serve as an efficient tool for analyzing GSH level in biosamples.

A label-free method for detecting biothiols based on poly(thymine)-templated copper nanoparticles.

A novel label-free nanosensor has been developed for detecting biothiols including cysteine, glutathione and homocysteine based on poly(thymine)-templated fluorescent copper nanoparticles (CuNPs), which were controlled through thymine-Hg(II)-thymine coordination. This assay provides a simple, cost-effective, and sensitive platform for the detection of biothiols. By employing this turn-on sensor, biological thiols, such as cysteine, glutathione and homocysteine, are successfully detected at concentrations as low as 12.5nM for cysteine, 15nM for glutathione, and 20nM for homocysteine. The sensing system also exhibits high selectivity against other amino acids, and the application of the sensor for biological fluids shows that the proposed method works well for real samples.