In-situ detection provides deep insights into the function of genes and their relationship with diseases by directly visualizing their spatiotemporal behavior. As an emerging in-situ imaging tool, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bioimaging can localize targets in living and fixed cells. CRISPR-mediated bioimaging has inherent advantages over the gold standard of fluorescent in-situ hybridization (FISH), including fast imaging, cost-effectiveness, and ease of preparation. Existing reviews have provided a detailed classification and overview of the principles of CRISPR-mediated bioimaging. However, the exploitation of potential clinical applicability of this bioimaging technique is still limited. Therefore, analyzing the potential value of CRISPR-mediated in-situ imaging is of great significance to the development of bioimaging. In this review, we initially discuss the available CRISPR-mediated imaging systems from the following aspects: summary of imaging substances, the design and optimization of bioimaging strategies, and factors influencing CRISPR-mediated in-situ detection. Subsequently, we highlight the potential of CRISPR-mediated bioimaging for application in biomedical research and clinical practice. Furthermore, we outline the current bottlenecks and future perspectives of CRISPR-based bioimaging. We believe that this review will facilitate the potential integration of bioimaging-related research with current clinical workflow.

Wearable biosensors monitoring various biomarkers in sweat provide comprehensive and prompt profiling of health states at molecular levels. Cytokines existed in sweat with trace amounts play an important role in cellular activity modulation. Unfortunately, flexible and wearable biosensors for cytokine monitoring have not yet been achieved due to the limitation of membrane-based structure and sensing strategy. Herein, we develop a novel electrochemical fabric based on aptamer-functionalized carbon nanotube/graphene fibers for real-time and in situ monitoring of IL-6, a paramount cytokine biomarker for inflammation and cancer. This fabric system possesses flexibility, anti-fatigue ability and breathability for wearable applications and can apply to different body parts in various forms. Moreover, the electrochemical fabric can track other biomarkers by replacing the coupling aptamer, serving as a universal platform for sweat analysis. This fabric-based platform holds the potential to facilitate an intelligent and personalized health monitoring approach.

A clinical diagnosis of chronic kidney disease (CKD) is commonly achieved by estimating the serum levels of urea and creatinine (CR). Given the limitations of the conventional diagnostic assays, it is imperative to seek alternative, economical strategies for the detection of CKD-specific biomarkers with high specificity and selectivity. In this respect, surface-enhanced Raman spectroscopy (SERS) can be regarded as an ideal choice. SERS signals can be greatly amplified by noble metal nanoparticles (e.g., gold nanoparticles (GNPs)) of numerous sizes, shapes, and configurations to help achieve ultra-sensitive single molecule-level detection at 10-15 M (up to 10 orders of magnitude more sensitive than fluorescence-based detection). The irregular geometry of GNPs with spike-like tips, dimers, and aggregates with small nanogaps (i.e., due to plasmon coupling such as Raman hot spots) play a pivotal role in enhancing the specificity and sensitivity of SERS. This review critically outlines the performance of SERS-based biosensors in the ultrasensitive detection of CKD biomarkers in various body fluids in terms of basic quality assurance parameters (e.g., limit of detection, figure of merit, enhancement factor, and stability of the biosensor). Moreover, the challenges and perspectives are described with respect to the expansion of such sensing techniques in practical clinical settings.

We demonstrate a smartphone integrated handheld (SPEED) digital polymerase chain reaction (dPCR) device for point-of-care application. The device has dimensions of ≈100 × 200 × 35 mm3 and a weight of ≈400 g. It can perform 45 PCR cycles in ≈49 min. The device also features integrated, miniaturized modules for thermal cycling, image taking, and wireless data communication. These functions are controlled by self-developed Android-based applications. The only consumable is the developed silicon-based dPCR chip, which has the potential to be recycled. The device's precision and accuracy are comparable with commercial dPCR machines. We have verified the SPEED dPCR prototype's utility in the testing of severe acute respiratory syndrome coronavirus 2, the detection of cancer-associated gene sequences, and the confirmations of Down syndrome diagnoses. Due to its low upfront capital investment, as well as its nominal running cost, we envision that the SPEED dPCR device will help to perform cancer screenings and non-invasive prenatal tests for the general population. It will also aid in the timely identification and monitoring of infectious disease testing, thereby expediting alerts with respect to potential emerging pandemics.

Allograft rejection has always been a major obstacle in organ transplantation. The current clinical diagnostic gold standard for allograft rejection is an invasive biopsy. However, biopsy has some limitations, such as sampling errors, risk of serious complications, and high cost. In this study, we have rationally developed an activatable fluorescent probe CYGB for imaging of granzyme B, which is a biomarker released by CD8+T cells attacking the graft. Moreover, the ability of CYGB to detect rejection early in mouse heart and skin transplantation models was evaluated. The probe CYGB consists of a caged hemicyanine-based fluorophore and a GzmB-specifically cleaved peptide substrate linked via a self-immolating spacer, p-aminobenzyl alcohol. Endogenous GzmB in CD8+ T cells specifically activated the near-infrared fluorescence (NIRF) signal of CYGB. In vivo imaging in mice skin and heart graft models, showed that CYGB preferentially accumulates in grafts, enabling early diagnosis of rejection. Moreover, CYGB enables non-invasive assessment of the level of immunosuppression in allogeneic mice treated with FK506. This study provides an alternative method for monitoring the status of allografts without biopsy.

Surface plasmon resonance (SPR)-based biosensors are widely used instruments for characterizing molecular interactions. In theory the SPR signal depends only on mass changes for interacting molecules of same chemical nature. Whether conformational changes of interacting molecules also contribute to the SPR signal is still a subject of lively debates. Works have been published claiming that conformational changes were detected but all factors contributing to the SPR signal were not carefully considered, in addition to often using no or improper controls. In the present work we used a very well-characterized oligonucleotide, the thrombin-binding DNA aptamer (TBA), which upon binding of potassium ions folds into a two G-tetrad antiparallel G-quadruplex structure. All terms contributing to the maximal expected SPR response, Rmax, in particular the refractive index increment, RII, of both partners and the fraction of immobilized TBA target available, ca, were experimentally assessed. The resulting Rmax was then compared to the maximal experimental SPR response for potassium ions binding to TBA using appropriate controls. Regardless how the RIIs were measured, by SPR or refractometry, and how much TBA available for interacting with potassium ions was considered, the theoretical and the experimental SPR responses never matched, the former being always lower than the latter. Using a straightforward experimental model system and by thoroughly taking into account all contributing factors we therefore conclude that conformational changes can indeed contribute to the measured SPR signal.

To achieve sensitive detection of low-content microRNA, photoelectrochemical/electrochromic dual-mode sensor with intrinsically low background signal has been developed, but the two detection modules are usually designed with a series-connected structure, which may cause signal interference and thus affect the detection reliability. To solve the above problems, a decoupled dual-mode bioassay for sensitive miRNA-21 detection with high reliability is constructed in this work, by selecting two capacitors to realize parallel amplification for the two detection modules, supplemented with a 3D DNA nanoring photoelectrode signal amplification strategy. The complete decoupling of the two detection modes, photoelectrochemical and electrochromic, as well as the use of digital multimeter, improves the reliability and accuracy of the sensor, and also frees it from dependence on electrochemical workstation, making detection more intuitive and faster. With simple structure, low cost, good reproducibility, high sensitivity, and easy operation, the capacitor-parallel-amplified decoupled photoelectrochemical/electrochromic dual-mode bioassay has broad application prospect in on-site point-of-care detection of diseases and low-cost clinical diagnosis. The design idea of decoupled dual-mode detector can also be extended to the construction of other dual-mode methods.

Circulating tumor cells (CTCs) as important biomarkers for noninvasive clinical diagnosis and prognostic evaluation are significant in predicting the overall survival and progression-free survival of cancer patients. However, the current typical CTCs separation and enrichment techniques were limited to a single collection of small-volume blood samples, which was inadequate to comprehensively profile the distribution of CTCs in the systemic blood. In addition, those techniques cannot reduce metastasis of CTCs unless adjuvant chemotherapy. Herein, inspired by hemodialysis, we designed a whole blood purifier (WBP) composed of a functionalized special spiral-like glass tube modified by anti-epithelial cell adhesion molecule (anti-EpCAM). The WBP allowed real-time capture, enrichment and removal of CTCs from systemic blood circulation, and the purified blood was immediately returned to the body. Furthermore, the WBP did not cause any organic damages in vivo. This approach achieves the high accuracy of liquid biopsy technology and is expected to become an effective clinical adjuvant therapy for tumor metastasis.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.

The state-of-art signal transduction mechanism of anodic photoelectrochemistry is constrained to the hole oxidation reaction, which greatly hinders its application for prospective biosensing applications. Herein, we present an innovative strategy for signal transduction by exploiting the in situ formation of surface oxygen vacancies (VOs) on Fe2O3 nanorods (NRs) through the self-coordination of 2,3-dihydroxynaphthalene (2,3-DHN) on their surfaces. The 2,3-DHN was connected with Fe(Ⅲ) on the surface of Fe2O3 NRs vis the formation of the five-membered ring structures accompanied by the generation of VOs. And the generated VOs introduced a new defect energy level for trapping the photogenerated holes, which enhanced the charge separation and realized the enhancement of photocurrent signal. The developed signal transduction strategy was validated by the first photoelectrochemical (PEC) sensing platform for β-glucoside (β-Glu) and lipase (LPS), which can catalyze the hydrolysis of 3-hydroxy-2-naphthalenyl-β-D-glucoside and naphthalene-2,3-diol diacetate, respectively, to produce 2,3-DHN for signal stimuli. The β-Glu and LPS were detected with linear ranges of 0.01-10.0 U/mL and 0.001-5.0 mg/mL, respectively. Detection limits of 3.3 × 10-3 U/mL and 0.32 μg/mL (S/N = 3) were achieved, for β-Glu and LPS, respectively. The present study not only provides a new strategy for spontaneous induction of VOs in situ for n-type semiconductors, but also innovates the anodic PEC signal transduction strategy with broadened biosensing applications.

Precise and early screening of colorectal cancer (CRC) is one crucial yet challenging task for its treatment, and the analysis of multi-targets of CRC in a single assay with high accuracy is essential for pathological research and clinical diagnosis. Here, a CRC-related biomarker pair, microRNA-211 (miRNA-211) and H2S, was detected by constructing a three-dimensional (3D) ordered DNA network. First, trace amount of miRNA-211 could initiate a hybridization chain reaction-based amplification process. A highly ordered 3D DNA network was formed based on the organized assembly of DNA-cube frameworks that were constructed by DNA origamis and Ag nanoparticles (NPs) encapsulated inside. In the presence of the H2S, Ag NPs within the network can be etched to generate Ag2S quantum dots, which could be better visualized in fluorescence in situ cell imaging. Using the 3D DNA ordered network as the sensing platform, it can acquire dual analysis of biomolecule (miRNA-211) and inorganic gas (H2S) in vitro, overcoming the limitations of single type of biomarker detection in a single assay. This assay achieved a wide linearity range of H2S from 0.05 to 10 μM, and exhibited a low limit of detection of 4.78 nM. This strategy allows us to acquire the spatial distributions of H2S and miRNA expression levels in living CRC cells simultaneously, providing a highly sensitive and selective tool for early screening and monitoring of CRC.

The high-throughput detection of multiple metabolites in saliva by electrochemical sensors is usually a challenge, which however is essential to the comprehensive evaluation of health status or screening of diseases. Here, a light-addressable and paper-based hydrogen peroxide (H2O2) photoelectrochemical (PEC) sensor for the high-throughput detection of multiple salivary metabolites is reported. This sensor has a unique solar microcell array structure with a silver nanowires/fullerene-Congo red (AgNWs/C60-CR) disc working electrode (WE) and a single-walled carbon nanotubes/platinum nanowires (SWCNTs/PtNWs) ring reference/counter electrode (RE/CE) in each microcell. Enzymes of different metabolites are immobilized on different separated microcells of a cover slide over the sensor, from which enzymatically produced H2O2 can react with p-hydroxyphenyl boric acid (4-HPBA) on the WE of the sensor to generate hydroquinone (HQ) for photocurrent responses. Based on this strategy, a disposable PEC sensor of saliva was developed, which allows the multiplexed detection of uric acid (UA), glucose (GLU) and lactate (LA) in diluted human saliva with high sensitivity and selectivity. Moreover, the detection throughput and application field of the sensor can be easily extended by connecting a series of sensors in parallel or varying the enzymes. The present work thus establishes a cost-effective approach to the scalable construction of versatile biosensing platforms with tunable throughput and varied analytes.

Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.

Rabies is a lethal zoonotic disease caused by rabies virus (RABV) that affects human health and the economy. RABV is transmitted mainly by bats in Brazil, and surveillance in remote areas is hampered by the difficulty of properly collecting samples during fieldwork and the diagnosis is performed in laboratory conditions. Here, we report a portable electrochemical biosensor based on nucleic acid interactions for RABV detection in nasopharyngeal swab samples. The working electrode of the biosensor is composed of reduced graphene oxide (rGO) thin-film immobilized with cDNA through pi-pi stacking to enhance virus detection and specificity. Sensor performance was determined using RNA, and swab samples from bats. RNA detection shows good selectivity, and quantification presents a highly linear calibration curve (R2 = 0.990) using a concentration range of 0.145-25.39 ng/μL. A LOD of 0.104 ng/μL was reached with a sensitivity of 0.321 μA (ng/μL)-1. RABV detection in nasopharyngeal swab samples showed a good difference of positive sample from negative with a response time in seconds, ultra-fast detection compared to known techniques. Three biosensor groups were identified and named after physic-chemical surface characterization as: GO-1, GO-2, and rGO; with best performance for rGO group due to its sp2 hybridized network. Thus, we have successfully fabricated a promising electrochemical biosensor for fast in-situ detection of the RABV in swab samples, which can be expanded to other enveloped viruses that have RNA.

Liquid crystal (LC)-based biosensors rely on the response of the LC molecules to perturbation generated by analytes at the interface, leading to the susceptible change in molecular alignment or orientation. The sensitivity of these biosensors is primarily dependent on the LC's material properties and surface anchoring strength. By incorporation of an unconventional mesogenic compound (CB7CB) coupled with the hybrid-alignment cell configuration, this work presents a binary nematic LC for label-free biosensing, manifesting a novel sensing technology that takes advantage of CB7CB-induced flexoelectricity in the transducer. Herein, we prepared LC mixtures by blending a typical rod-like nematic LC (E7) with the bent-core mesogen CB7CB in various weight ratios and studied the effect of the CB7CB content on E7/CB7CB-based biosensing performance in vertically aligned and hybrid-aligned nematic (HAN) cells. Owing to the anomalously small bend elastic constant K33 in CB7CB, the mixture designated CB45 with the highest CB7CB weight percentage (45 wt% in this study) was best applicable to biosensing in HAN cells. When observed under a polarizing optical microscope, CB45 in the HAN geometry showed the capability of detection of as low as 10-10 g/mL for the protein standard bovine serum albumin (BSA). Moreover, the quantitation of the assay was fulfilled by both dielectric and light transmission measurements of the hybrid-aligned cholesteric CB45/R5011. The limit of detection of 7 × 10-10 g/mL was achieved by spectrometric analysis. To the best of our knowledge, this work is the first to demonstrate flexoelectric biosensing on the basis of flexoelectric polarization associated with giant flexoelectricity in CB7CB partially constituting the LC transducer.

In this work, a unique FeMoOv nanozyme-bipolar electrode (NM-BPE) electrochemiluminescence (ECL) biosensing and imaging platform was proposed for the first time to realize sensitive detection of target hydrogen peroxide (H2O2) and prostate specific antigen (PSA). Considering the advantage that the cathode and anode poles of the bipolar electrode (BPE) can be modified respectively, this work was carried out using anode equipped with ECL reagent bipyridine ruthenium (Ru(bpy)32+), and cathode equipped with the Fe-doped molybdenum oxide/Au nanoparticles (FeMoOv/AuNPs) with excellent peroxidase (POD) and catalase (CAT)-like activity. Because FeMoOv/AuNPs show efficient enzyme catalysis effect and can greatly promote the decomposition of H2O2, thus the electron transfer rate in the NM-BPE system would be much accelerated to enhance the ECL signal of Ru(bpy)32+. Based on this principle, this work not only realized sensitive detection of H2O2, but also ingeniously designed an sandwich immunosensor using FeMoOv/AuNPs as recognition probe to mediate the ECL response on the anode, achieving highly sensitive detection of PSA. Furthermore, a unique mobile phone ECL imaging system was developed for assay of PSA at different concentrations, which opened a new portable imaging sensing device for bioassays. This work was the first time to combine nanozymes with bipolar electrodes for ECL analysis and imaging, which not only broadened the applications of nanozymes, but also pioneered the new joint ECL research technique of bipolar electrode and ECL imaging in bioassays, showing great application prospect for multiple detection of proteins, nucleic acids and cancer cells.

As a "star molecule", nitric oxide (NO) either promotes or inhibits many physiological processes depending on its concentration. The in situ generation and monitoring of therapeutic gas molecules has been a problem that many researchers have been working to address due to the stochastic nature of gas molecule movement. There are still relatively few studies using short peptides as NO storage systems, and there are still challenges in monitoring NO release in situ with real-time imaging over long periods of time. In this work, a morphologically transformable NO release, diagnosis and treatment integrated multifunctional nanoplatform was fabricated. A new NO-activated probe (DPBTD) with emission in the first near infrared (NIR-I) region was encapsulated into the hydrophobic domains of Ac-KLVFFAL-NH2 peptide derivatives as a biosensor for NO release. Peptide scaffolds were endowed with the capacity of controlled NO release by the introduction of NO donor (organic nitrates). Interestingly, morphology of the nanoplatform could be transformed from one-dimensional (1D) nanowires to two-dimensional (2D) nanosheets via nanorods transition state under tip sonication, which was allowed for better cell uptake. Eventually, this nanocarrier was used for stimuli-responsive NO release, real-time imaging and treatment in tumor tissues of 4T1 tumor-bearing mice. This strategy expands the application potential of peptide-based nanomaterials and provides ideas for monitoring the progress of gas-mediated cancer therapy.

A photoelectrochemical (PEC) aptasensor for thrombin detection was rationally designed based on the photoanode of one-dimensional hematite nanorods (α-Fe2O3 NRs) with several steps of modifications. Uniform α-Fe2O3 NRs were grown vertically on the surface of fluorine-doped tin oxide (FTO) conductive glass through a one-step hydrothermal method; then Ag was grown on the surface of α-Fe2O3 NRs through a photoreduction method followed by a partial in-situ transformation into Ag2S, conferring an improvement on the initial photocurrent. Two main critical factors, namely, the steric hindrance of thrombin, benzoquinone (BQ) precipitation oxidized by H2O2 under the catalysis of G-quadruplexes/hemin, contributed to the sensitive signal-down response toward the target. Photocurrent signals related with thrombin concentration was established for thrombin analysis due to the non-conductive complex as well as their competitive consumption of electron donors and irradiation light. The excellent initial photocurrent was combined with the signal-down amplification in the design of the biosensor, conferring a limit of detection (LOD) as low as 40.2 fM and a wide linear range from 0.0001 nM to 50 nM for the detection of thrombin. The proposed biosensor was also assessed in terms of selectivity, stability, and applicability in human serum analyses, which provided an appealing maneuver for the specific analysis of thrombin in trace amount.

Current in-vitro 2D cultures and animal models present severe limitations in recapitulating human physiopathology with striking discrepancies in estimating drug efficacy and side effects when compared to human trials. For these reasons, microphysiological systems, organ-on-chip and multiorgans microdevices attracted considerable attention as novel tools for high-throughput and high-content research to achieve an improved understanding of diseases and to accelerate the drug development process towards more precise and eventually personalized standards. This review takes the form of a guide on this fast-growing field, providing useful introduction to major themes and indications for further readings. We start analyzing Organs-on-chips (OOC) technologies for testing the major drug administration routes: (1) oral/rectal route by intestine-on-a-chip, (2) inhalation by lung-on-a-chip, (3) transdermal by skin-on-a-chip and (4) intravenous through vascularization models, considering how drugs penetrate in the bloodstream and are conveyed to their targets. Then, we focus on OOC models for (other) specific organs and diseases: (1) neurodegenerative diseases with brain models and blood brain barriers, (2) tumor models including their vascularization, organoids/spheroids, engineering and screening of antitumor drugs, (3) liver/kidney on chips and multiorgan models for gastrointestinal diseases and metabolic assessment of drugs and (4) biomechanical systems recapitulating heart, muscles and bones structures and related diseases. Successively, we discuss technologies and materials for organ on chips, analyzing (1) microfluidic tools for organs-on-chips, (2) sensor integration for real-time monitoring, (3) materials and (4) cell lines for organs on chips. (Nano)delivery approaches for therapeutics and their on chip assessment are also described. Finally, we conclude with a critical discussion on current significance/relevance, trends, limitations, challenges and future prospects in terms of revolutionary impact on biomedical research, preclinical models and drug development.

As a full-fidelity simulation of human cells, tissues, organs, and even systems at the microscopic scale, Organ-on-a-Chip (OOC) has significant ethical advantages and development potential compared to animal experiments. The need for the design of new drug high-throughput screening platforms and the mechanistic study of human tissues/organs under pathological conditions, the evolving advances in 3D cell biology and engineering, etc., have promoted the updating of technologies in this field, such as the iteration of chip materials and 3D printing, which in turn facilitate the connection of complex multi-organs-on-chips for simulation and the further development of technology-composite new drug high-throughput screening platforms. As the most critical part of organ-on-a-chip design and practical application, verifying the success of organ model modeling, i.e., evaluating various biochemical and physical parameters in OOC devices, is crucial. Therefore, this paper provides a logical and comprehensive review and discussion of the advances in organ-on-a-chip detection and evaluation technologies from a broad perspective, covering the directions of tissue engineering scaffolds, microenvironment, single/multi-organ function, and stimulus-based evaluation, and provides a more comprehensive review of the progress in the significant organ-on-a-chip research areas in the physiological state.

Plant stress responses involve a suite of genetically encoded mechanisms triggered by real-time interactions with their surrounding environment. Although sophisticated regulatory networks maintain proper homeostasis to prevent damage, the tolerance thresholds to these stresses vary significantly among organisms. Current plant phenotyping techniques and observables must be better suited to characterize the real-time metabolic response to stresses. This impedes practical agronomic intervention to avoid irreversible damage and limits our ability to breed improved plant organisms. Here, we introduce a sensitive, wearable electrochemical glucose-selective sensing platform that addresses these problems. Glucose is a primary plant metabolite, a source of energy produced during photosynthesis, and a critical molecular modulator of various cellular processes ranging from germination to senescence. The wearable-like technology integrates a reverse iontophoresis glucose extraction capability with an enzymatic glucose biosensor that offers a sensitivity of 22.7 nA/(μM·cm2), a limit of detection (LOD) of 9.4 μM, and a limit of quantification (LOQ) of 28.5 μM. The system's performance was validated by subjecting three different plant models (sweet pepper, gerbera, and romaine lettuce) to low-light and low-high temperature stresses and demonstrating critical differential physiological responses associated with their glucose metabolism. This technology enables non-invasive, non-destructive, real-time, in-situ, and in-vivo identification of early stress response in plants and provides a unique tool for timely agronomic management of crops and improving breeding strategies based on the dynamics of genome-metabolome-phenome relationships.

Early screening of biomarkers benefits therapy and prognosis of cancers. MiRNAs encapsulated in tumor-derived exosomes are emerging biomarkers for early diagnosis of cancers. Nevertheless, traditional methods suffer certain drawbacks, which hamper their wide applications. In this contribution, we have developed a convenient electrochemical approach for quantification of exosomal miRNA based on the assembly of DNA triangular pyramid frustum (TPF) and strand displacement amplification. Four single-stranded DNA helps the formation of primary DNA triangle with three thiols for gold electrode immobilization at the bottom and three amino groups on overhangs for the capture of silver nanoparticles. On the other hand, target miRNA induced strand displacement reaction produces abundant specific DNA strands, which help the DNA structural transition from triangle to TPF. Amino groups are thus hidden and the declined silver stripping current can be used for the evaluation of target miRNA concentration. This biosensor exhibits excellent analytical performances and successfully achieves analysis of exosomal miRNAs from cells and clinical serum samples.

Bacterial spores are problematic in agriculture, the food industry, and healthcare, with the fallout costs from spore-related contamination being very high. Spores are difficult to detect since they are resistant to many of the bacterial disruption techniques used to bring out the biomarkers necessary for detection. Because of this, effective and practical spore disruption methods are desirable. In this study, we demonstrate the efficiency of a compact microfluidic lab-on-chip built around a coplanar waveguide (CPW) operating at 2.45 GHz. We show that the CPW generates an electric field hotspot of ∼10 kV/m, comparable to that of a commercial microwave oven, while using only 1.2 W of input power and thus resulting in negligible sample heating. Spores passing through the microfluidic channel are disrupted by the electric field and release calcium dipicolinic acid (CaDPA), a biomarker molecule present alongside DNA in the spore core. We show that it is possible to detect this disruption in a bulk spore suspension using fluorescence spectroscopy. We then use laser tweezers Raman spectroscopy (LTRS) to show the loss of CaDPA on an individual spore level and that the loss increases with irradiation power. Only 22% of the spores contain CaDPA after exposure to 1.2 W input power, compared to 71% of the untreated control spores. Additionally, spores exposed to microwaves appear visibly disrupted when imaged using scanning electron microscopy (SEM). Overall, this study shows the advantages of using a CPW for disrupting spores for biomarker release and detection.

Misuse and overuse of tetracycline antibiotics (TCs) brings serious issues to ecological environment, food safety and human health. It is urgent to develop unique platform for high efficient identification and removal of TCs. In the present investigation, an effective and simple fluorescence sensor array was constructed based on the interaction between metal ions (Eu3+ and Al3+) and antibiotics. Benefiting from the different affinities between the ions and TCs, the sensor array can identify TCs from other antibiotics, which also can further differentiating four kinds of TCs (OTC, CTC, TC and DOX) from each other via linear discriminant analysis (LDA) technique. Meanwhile, the sensor array performed well in quantitative analysis of single TC antibiotic and differentiation of TCs mixtures. More interestingly, Eu3+ and Al3+-doped sodium alginate/polyvinyl alcohol hydrogel beads (SA/Eu/PVA and SA/Al/PVA) were further constructed, which can not only identify the TCs but simultaneously remove the antibiotics with high efficiency. The investigation provided an instructive way for rapid detection and environment protection.

Sensitive detection of metoprolol tartrate (MPT) is extremely urgent in the therapy of cardiovascular diseases to guarantee the curative effectiveness. Herein, porous ZrO2 was first employed as a matrix to spatially confine CuNCs (ZrO2@CuNCs), which simultaneously ameliorated the emission intensity and stability of CuNCs. Benefiting from the inner filter effect (IFE) and dynamic quenching effect (DQE) between ZrO2@CuNCs and AuNPs and the color fading of AuNPs induced by MPT, fluorometric and colorimetric methods for simple and sensitive determination of MPT were proposed. Besides, to meet the demand of convenient detection of MPT, a portable sensing platform was constructed including a dark box produced by a 3D printer and a smartphone. This method was further employed to determine MPT in human serum and urine samples with satisfactory results with the triple mode. This work is the first attempt to fabricate a multi-mode optical and portable sensor for MPT detection, which provides a novel approach for point-of-care monitoring of drugs in the treatment of diseases.

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. There is a need for rapid diagnostic tests, ideally at point-of-care. MicroRNA 122 (miR-122) is an early biomarker for DILI which is reported to increase in the blood before standard-of-care markers such as alanine aminotransferase activity. We developed an electrochemical biosensor for diagnosis of DILI by detecting miR-122 from clinical samples. We used electrochemical impedance spectroscopy (EIS) for direct, amplification free detection of miR-122 with screen-printed electrodes functionalised with sequence specific peptide nucleic acid (PNA) probes. We studied the probe functionalisation using atomic force microscopy and performed elemental and electrochemical characterisations. To enhance the assay performance and minimise sample volume requirements, we designed and characterised a closed-loop microfluidic system. We presented the EIS assay's specificity for wild-type miR-122 over non-complementary and single nucleotide mismatch targets. We successfully demonstrated a detection limit of 50 pM for miR-122. Assay performance could be extended to real samples; it displayed high selectivity for liver (miR-122 high) comparing to kidney (miR-122 low) derived samples extracted from murine tissue. Finally, we successfully performed an evaluation with 26 clinical samples. Using EIS, DILI patients were distinguished from healthy controls with a ROC-AUC of 0.77, a comparable performance to qPCR detection of miR-122 (ROC-AUC: 0.83). In conclusion, direct, amplification free detection of miR-122 using EIS was achievable at clinically relevant concentrations and in clinical samples. Future work will focus on realising a full sample-to-answer system which can be deployed for point-of-care testing.

Bacterial cellulose (BC) with its inherent nanofibrils framework is an attractive building block for the fabrication of sustainable bioelectronics, but there still lacks an effective and green strategy to regulate the hydrogen-bonding topological structure of BC to improve its optical transparency and mechanical stretchability. Herein, we report an ultra-fine nanofibril-reinforced composite hydrogel by utilizing gelatin and glycerol as hydrogen-bonding donor/acceptor to mediate the rearrangement of the hydrogen-bonding topological structure of BC. Attributing to the hydrogen-bonding structural transition, the ultra-fine nanofibrils were extracted from the original BC nanofibrils, which reduced the light scattering and endowed the hydrogel with high transparency. Meanwhile, the extracted nanofibrils were connected with gelatin and glycerol to establish an effective energy dissipation network, leading to an increase in stretchability and toughness of hydrogels. The hydrogel also displayed tissue-adhesiveness and long-lasting water-retaining capacity, which acted as bio-electronic skin to stably acquire the electrophysiological signals and external stimuli even after the hydrogel was exposing to air condition for 30 days. Moreover, the transparent hydrogel could also serve as a smart skin dressing for optical identification of bacterial infection and on-demand antibacterial therapy after combined with phenol red and indocyanine green. This work offers a strategy to regulate the hierarchical structure of natural materials for designing skin-like bioelectronics toward green, low cost, and sustainability.

Cell death resulting from ferroptosis is a consequence of the accumulation of lipid peroxides that are produced when lipids and reactive oxygen species (ROS) interact. This process is dependent on iron and alters the structure and polarity of lipid droplets (LDs). Unlike reactive fluorescent probes, environment-sensitive fluorescent probes can accurately monitor metabolic activities by sensing the intracellular environment of living organisms. To this end, we developed a polarity-sensitive fluorescent probe LIP-Ser that anchors to LDs and can be used to monitor changes in the polarity of LDs during ferroptosis by in situ imaging. LIP-Ser has a red-emitting (λem = 634 nm) and a large Stokes shift (Δλ = 161 nm in 1,4-dioxane), which avoids it from autofluorescence interference and crosstalk between excitation and emission spectra, thereby preventing low signal-to-noise ratio and severe fluorescence self-quenching during imaging. Additionally, LIP-Ser is used in this study to demonstrate that non-alcoholic fatty liver disease (NAFLD) promotes ferroptosis at the cellular and in vivo levels, and that inhibition of cellular ferroptosis effectively reduces the damage caused by NAFLD to cells and mouse liver tissue.

Nondestructive separation/enrichment and reliable detection of extremely rare circulating tumor cells (CTCs) in peripheral blood are of considerable importance in tumor precision diagnosis and treatment, yet this remains a big challenge. Herein, a novel strategy for nondestructive separation/enrichment and ultra-sensitive surface-enhanced Raman scattering (SERS)-based enumeration of CTCs is proposed via aptamer recognition and rolling circle amplification (RCA). In this work the magnetic beads modified with "Aptamer (Apt)-Primer" (AP) probes were utilized to specifically capture CTCs, and then after magnetic separation/enrichment, the RCA-powered SERS counting and benzonase nuclease cleavage-assisted nondestructive release of CTCs were realized, respectively. The AP was assembled by hybridizing the EpCAM-specific aptamer with a primer, and the optimal AP contains 4 mismatched bases. The RCA enhanced SERS signal nearly 4.5-fold, and the SERS strategy has good specificity, uniformity and reproducibility. The proposed SERS detection possesses a good linear relationship with the concentration of MCF-7 cells spiked in PBS with the limit of detection (LOD) of 2 cells/mL, which shows good potential practicality for detecting CTCs in blood with recoveries ranging from 100.56% to 116.78%. Besides, the released CTCs remained good cellular activity with the normal proliferation after re-culture for 48 h and normal growth for at least three generations. The proposed strategy of nondestructive separation/enrichment and SERS-based sensitive enumeration is promising for reliable analysis of EpCAM-positive CTCs in blood, which is expected to provide a powerful tool for analysis of extremely rare circulating tumor cells in complex peripheral blood for liquid biopsy.

The circulating tumor DNA (ctDNA) is a crucial cancer marker, its sensitive monitoring is useful for early diagnose and therapy of tumor-related diseases. Herein, a bipedal DNA walker with multiple recognition sites is designed through the transition of dumbbell-shaped DNA nanostructure to realize the dual amplification of the signal and achieve ultrasensitive photoelectrochemical (PEC) detection of ctDNA. Initially, the ZnIn2S4@AuNPs is obtained by combining the drop coating method with electrodeposition method. When the target is present, the dumbbell-shaped DNA structure transforms into an annular bipedal DNA walker that can walk unrestrictedly on the modified electrode. After the cleavage endonuclease (Nb.BbvCI) was added to the sensing system, the ferrocene (Fc) on the substrate is released from the electrode surface, and the transfer efficiency of photogenerated electron-hole pairs is extremely improved, enabling the "signal on" testing of ctDNA. The detection limit of the prepared PEC sensor is 0.31 fM, and the recovery of actual samples varied between 96.8 and 103.6% with an average relative standard deviation of about 8%. Meaningfully, the prepared PEC biosensor with an innovative bipedal DNA walker has potential application value for ultrasensitive detection of other nucleic acid-related biomarker.

The organic luminophores have inspired widespread interest in electrochemiluminescence (ECL). Herein, a novel rod-like metal-organic framework was formed by chelating Zn ion with 9,10-di(p-carboxyphenyl)-anthracene (DPA), defined as Zn-MOF for simplicity. In this proposal, the prepared Zn-MOF was first used as a powerful organic luminophore with low trigger potential, thus developing a competitive ECL immunoassay for ultrasensitive detection of 5-fluorouracil (5-FU) with 1,4-diazabicyclo[2.2.2]octane (D-H2) as the coreactant. The absorption spectrum of cobalt oxyhydroxide (CoOOH) nanosheets and the ECL emission spectrum of Zn-MOF could be highly matched, which ensured the occurrence of resonance energy transfer (RET). For that, ECL-RET was applied in the assembly strategy of the ECL biosensor, and Zn-MOF was used as the energy donor and CoOOH nanosheets as the acceptor. Taking advantage of the luminophore and ECL-RET, the immunoassay can be used for ultra-sensitive quantitative detection of 5-fluorouracil. The proposed ECL-RET immunosensor showed satisfactory sensitivity and accuracy with a wider linear range from 0.001 to 1000 ng/mL, and a lower detection limit (0.52 pg/mL). Hence, it is worth believing that this strategy can pave a bright research direction for the detection of 5-FU or other biological small molecules.

Natriuretic peptides can relieve cardiovascular stress and closely related to heart failure. Besides, these peptides also have preferable interactions of binding to cellular protein receptors, and subsequently mediate various physiology actions. Hence, detection of these circulating biomarkers could be evaluated as a predictor ("Gold standard") for rapid, early diagnosis and risk stratification in heart failure. Herein, we proposed a measurement to discriminate multiple natriuretic peptides via the peptide-protein nanopore interaction. The nanopore single-molecular kinetics revealed that the strength of peptide-protein interactions was in the order of ANP > CNP > BNP, which was demonstrated by the simulated peptide structures using SWISS-MODEL. More importantly, the peptide-protein interaction analyzing also allowed us to measure the peptide linear analogs and structure damage in peptide by single-chemical bond breakup. Finally, we presented an ultra-sensitive detection of plasma natriuretic peptide using asymmetric electrolyte assay, obtaining a detection limit of ∼770 fM for BNP. At approximately, it is 1597 times lower than that of using symmetric assay (∼1.23 nM), 8 times lower than normal human level (∼6 pM), and 13 times lower than the diagnostic values (∼10.09 pM) complied in the guideline of European Society of Cardiology. That said, the designed nanopore sensor is benefit for natriuretic peptides measurement at single molecule level and demonstrates its potential for heart failure diagnosis.

Chemotherapy failure in pancreatic cancer patients is widely attributed to cancer cell reprogramming towards drug resistance by cancer associated fibroblasts (CAFs), which are the abundant cell type in the tumor microenvironment. Association of drug resistance to specific cancer cell phenotypes within multicellular tumors can advance isolation protocols for enabling cell-type specific gene expression markers to identify drug resistance. This requires the distinction of drug resistant cancer cells versus CAFs, which is challenging since permeabilization of CAF cells during drug treatment can cause non-specific uptake of cancer cell-specific stains. Cellular biophysical metrics, on the other hand, can provide multiparametric information to assess the gradual alteration of target cancer cells towards drug resistance, but these phenotypes need to be distinguished versus CAFs. Using pancreatic cancer cells and CAFs from a metastatic patient-derived tumor that exhibits cancer cell drug resistance under CAF co-culture, the biophysical metrics from multifrequency single-cell impedance cytometry are utilized for distinction of the subpopulation of viable cancer cells versus CAFs, before and after gemcitabine treatment. This is accomplished through supervised machine learning after training the model using key impedance metrics for cancer cells and CAFs from transwell co-cultures, so that an optimized classifier model can recognize each cell type and predict their respective proportions in multicellular tumor samples, before and after gemcitabine treatment, as validated by their confusion matrix and flow cytometry assays. In this manner, an aggregate of the distinguishing biophysical metrics of viable cancer cells after gemcitabine treatment in co-cultures with CAFs can be used in longitudinal studies, to classify and isolate the drug resistant subpopulation for identifying markers.