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Digital immunoassays enable the detection of protein biomarkers with very low concentrations, but the analysis stringently requires single-bead encapsulation. Low bead density has been adopted to minimize multiple-bead encapsulations, but the trade-off is the low droplet effectiveness (∼10%) in droplet-based assays. Here we report the method of inclusive droplet digital ELISA (iddELISA) that embraces all types of encapsulations by factoring in their varied “on–off” probabilities in the statistical inference. We derived the statistical model, optimized the bead encapsulation and immunoreaction, and developed an image analysis pipeline for accurate droplet and bead recognition, showing that approximately 40% of the droplets could be used in the analysis. Using the detection of SARS-CoV-2 nucleocapsid protein as a demonstration, the iddELISA achieved a limit of detection of 0.71 fg/mL, which was much lower than conventional ELISA as well as droplet digital ELISA. By effectively incorporating multiple bead encapsulations, the iddELISA simplified the digital immunoassay while improving the counting efficiency and sensitivity, representing a unique concept in digital immunoassays.

H2S is a gaseous signaling molecule that is involved in many physiological and pathological processes. In general, the level of intracellular H2S (<1 μM) is much lower than that of GSH (∼1–10 mM), leading to the remaining challenge of selective detection and differentiation of endogenous H2S in live biosystems. To this end, we quantitatively demonstrate that the thiolysis of NBD amine has much higher selectivity for H2S over GSH than that of the reduction of aryl azide. Subsequently, we developed the first NBD-based cell-trappable probe 1(AM-BODIPY-NBD) for highly selective and ultrasensitive imaging of intracellular H2S. Probe 1demonstrates a 207-fold fluorescence enhancement at 520 nm after reaction with H2S/esterase to produce a bright BODIPY (quantum yield 0.42) and a detection limit of 15.7 nM. Probe 1is water-soluble, cell-trappable, and not cytotoxic. Based on this excellent chemical tool, relative levels of basal H2S in different cell lines were measured to reveal a positive correlation between endogenous H2S and the metastatic potential of colon and breast cancer cells. In addition, H2S biogenesis in vivowas also validated by probe 1both in tobacco leaves under viral infection and in zebrafish after tail amputation. It is anticipated that probe 1will have widespread applications in imaging and for investigating different H2S-related biological processes and diseases.

Albeit frequently being overlooked, MS2spectrum variation against collision energy (CE) implies auxiliary structural clues for m/zvalues. Online energy-resolved MS (ER-MS) provides the opportunity to acquire the trajectory of ion intensity against CE for any fragment ion of interest, thus exactly offering the desired momentum to empower the conventional MS2spectrum at a certain CE forward to a full-CE ramp MS2spectrum (FCER-MS2). Efforts were made here to construct an FCER-MS2spectrum and to evaluate its potential toward structural analysis. Flavonoids were employed as a proof of concept. MS2spectra of 76 compounds were recorded by LC–Q-Exactive-MS, and online ER-MS was subsequently programmed using LC–Qtrap-MS to build a breakdown graph for each obvious fragment ion. After defining the greatest value amongst all regressive apices as 100%, the normalized breakdown graphs comprised an FCER-MS2spectrum for each compound. The FCER-MS2spectrum contained the MS2spectrum at any CE as well as optimal CE (OCE) and maximal relative ion intensity (RIImax) of each fragment ion. Except the pronounced isomeric discrimination potential, either OCE or RIImaxreflected certain structural properties, such as aglycone, glycosidic bond, and hydroxy, methoxy, and glycosyl substituents. These rules were subsequently applied for flavonoid-focused characterization of a famous herbal medicine, namely Scutellariae Radix, and high-level structural annotation was accomplished for 75 flavonoids. Above all, the FCER-MS2spectrum includes m/z, OCEs, and RIImaxfeatures, thus facilitating confidence-advanced structural analysis.

A microfluidic paper-based analytical device (μPAD) is a cost-effective platform to implement assays, especially for point-of-care testing. Developing μPADs with fluidic control is important to implement multistep assays and provide high sensitivities. However, current localized delays in μPADs made of sucrose have a limited ability to decrease the flow rate. In addition, existing μPADs for automatic multistep assays are limited by their need for auxiliary instruments, their false activation, or their unavoidable tradeoff between available fluid volumes and temporal differences between steps. Here, a novel μPAD composed of a localized dissolvable delay and a horizontal motion mechanical valve for use as an automatic multistep assay is reported. A mixture of fructose and sucrose was used in the localized dissolvable delay and it provided an effective decrease in the flow rate to ensure adequate sensitivity in an assay. The dissolvable delay effectively doubled the flow time. A mechanical valve using a horizontal movement was developed to automatically implement a multistep process. Two-step and four-step processes were enabled with the μPAD. Cardiac troponin I (cTnI), a gold-standard biomarker for myocardial infarction, was used as a model analyte to show the performance of the developed μPAD in an assay. The designed μPAD, with the simple-to-make localized dissolvable delay and the robust mechanical valve, provides the potential to automatically implement high-performance multistep assays toward a versatile platform for point-of-care diagnostics.

Alzheimer’s disease (AD) is the most common neurodegenerative disease among the elderly. Abnormal aggregates of both β-amyloid peptide (Aβ) subtypes, Aβ42and Aβ40, are the typical neuropathology hallmarks of AD. However, because of the lack of specific recognition elements such as an antibody and aptamer, it is difficult to differentiate and determine the oligomers of Aβ42and Aβ40in clinic. In this paper, we developed a planar bilayer lipid membrane (BLM)-based electrochemical biosensor. According to the dynamic differences on oligomer-induced BLM damage, both low-mass, soluble oligomers of Aβ42and Aβ40(L-Aβ42O and L-Aβ40O) were measured in turn by electrochemical impedance spectroscopy. The BLM was supported by a porous 11-mercaptoundecanoic acid layer on a gold electrode, which amplified the impedance signal corresponding to the membrane damage and improved the detection sensitivity. The weakly charged surface of the BLM ensured the low non-specific adsorption of coexisting proteins in cerebrospinal fluid (CSF). Using the electrochemical biosensor, L-Aβ42O was determined within 20 min, with a linear range from 5 to 500 pM and a detection limit of 3 pM. Meanwhile, L-Aβ40O was determined within 60 min, with a linear range from 60 pM to 6.0 nM and a detection limit of 26 pM. The recoveries in oligomer-spiked artificial CSF and human CSF samples confirmed the accuracy and applicability of this proposed method in clinic. This work provides an antibody-free, highly selective, and sensitive method for simultaneous detections of L-Aβ42O and L-Aβ40O in real CSF samples, which is significant for the early diagnosis and prognosis of AD.

Direct infusion-mass spectrometry (DI-MS) currently serves as an alternative analytical tool for metabolomics owing to the unique high-throughput advantage. Except the inherent shortcoming at a significant matrix effect, there are two other primary technical obstacles dampening its wide applications, such as data alignment and structural annotation. To address these two obstacles, a novel strategy termed as DI-three-dimensional-MS (DI-3D-MS) was proposed here, and chemome comparison among several confusing herbal medicines (HMs) belonging to the Umbelliferaefamily was conducted as a proof-of-concept. Each test sample was directly infused into Qtrap-MS. In the first dimension, stepwise multiple ion monitoring (MIM) program was implemented to universally acquire the quantitative information on all HMs and to generate aligned data files. In the second dimension, MS2spectra were universally recorded by enhanced product ion (EPI) experiments that were triggered by MIM via an information-dependent acquisition algorithm. In the third dimension, online energy-resolved MS (ER-MS) was programmed to yield breakdown graphs for all MIM items. Moreover, a data library was built to aid structural identification by involving MS2and CE50features that were obtained by well-developed LC–MS methods. Qualitative and quantitative potentials of DI-3D-MS were validated toward metabolomics study. Significant species differences were observed, and all materials were grouped into three clusters. After matching MS2spectra and breakdown graphs between DI-3D-MS and those in the data library, coumarins ubiquitously existed in each HM, and angular-type pyranocoumarins, linear-type pyranocoumarins, angular-type furanocoumarins, along with ligustilide derivatives offered primary contributions for the classification pattern. Above all, DI-3D-MS is an eligible choice for rapid metabolomics of HMs and other matrices as well.

Exocytosis and retrieval of synaptic vesicles (SVs) are vital steps during neurotransmitter propagation between neurons. Visualization of this dynamics of SVs is significant for elucidating the mechanisms underlying synaptic transmission but remains challenging without an efficient, reliable, and biocompatible labeling method. In this work, we developed pH-responsive ratiometric DNA tetrahedral nanoprobes (pHadtnps) that could specifically label recycling SVs with high stability and effective background suppression. On the basis of the luminal pH alternation during the recycling of SVs, pHadtnps were able to illustrate their exocytosis and retrieval in real time. Moreover, with the high programmability of DNA nanotechnology, these nanoprobes could be flexibly equipped with different functional moieties, holding great promise for developing various versatile tools for studying communication in neuronal networks.