Lipids are important molecules that are widely distributed within the cell, and they play a crucial role in several biological processes such as cell membrane formation, signaling, cell motility and division. Monitoring the spatiotemporal dynamics of cellular lipids in real-time and quantifying their concentrations in situ is crucial since the local concentration of lipids initiates various signaling pathways that regulate cellular processes. In this review, we first introduced the historical background of lipid quantification methods. We then delve into the current state of the art of in situ lipid quantification, including the establishment and utility of fluorescence imaging techniques based on sensors of lipid-binding domains labeled with organic dyes or fluorescent proteins, and Raman and magnetic resonance imaging (MRI) techniques that do not require lipid labeling. Next, we highlighted the biological applications of live-cell lipid quantification techniques in the study of in situ lipid distribution, lipid transformation, and lipid-mediated signaling pathways. Finally, we discussed the technical challenges and prospects for the development of lipid quantification in live cells, with the aim of promoting the development of in situ lipid quantification in live cells, which may have a profound impact on the biological and medical fields.

The tremendous technical progress in neuroscience offers opportunities to observe a more minor or/and broader dynamic picture of the brain. Moreover, the large-scale neural activity of individual neurons enables the dissection of detailed mechanistic links between neural populations and behaviors. To measure neural activity in-vivo, multi-neuron recording, and neuroimaging techniques are employed and developed to acquire more neurons. The tools introduced concurrently recorded dozens to hundreds of neurons in the coordinated brain regions and elucidated the neuronal ensembles from a massive population perspective of diverse neurons at cellular resolution. In particular, the increasing spatiotemporal resolution of neuronal monitoring across the whole brain dramatically facilitates our understanding of additional nervous system functions in health and disease. Here, we will introduce state-of-the-art neuroscience tools involving large-scale neural population recording and the long-range connections spanning multiple brain regions. Their synergic effects provide to clarify the controversial circuitry underlying neuroscience. These challenging neural tools present a promising outlook for the fundamental dynamic interplay across levels of synaptic cellular, circuit organization, and brain-wide. Hence, more observations of neural dynamics will provide more clues to elucidate brain functions and push forward innovative technology at the intersection of neural engineering disciplines. We hope this review will provide insight into the use or development of recent neural techniques considering spatiotemporal scales of brain observation.

Lipopolysaccharides (LPS) are the major constituent on the cell envelope of all gram-negative bacteria. They are ubiquitous in air, and are toxic inflammatory stimulators for urinary disorders and sepsis. The reported optical, thermal, and electrochemical sensors via the intermolecular interplay of LPS with proteins and aptamers are generally complicated methods. We demonstrate the single-molecule nanopore approach for LPS identification in distinct bacteria as well as the serotypes discrimination. With a 4 nm nanopore, we achieve a detection limit of 10 ng/mL. Both the antibiotic polymyxin B (PMB) and DNA aptamer display specific binding to LPS. The identification of LPS in both human serum and tap water show good performance with nanopore platforms. Our work shows a highly-sensitive and easy-to-handle scheme for clinical and environmental biomarkers determination and provides a promising screening tool for early warning of contamination in water and medical supplies.

Diabetes is known to cause a variety of complications, having a high correlation with Alzheimer's disease. Electrophysiological recording using a microscale needle electrode is a promising technology for the study, however, diabetic brain tissue is more difficult to record neuronal activities than normal tissue due to these complications including the development of cerebrovascular disease. Here we show an electrophysiological methodology for diabetic db/db mice (+Leprdb/+Leprdb) using a 4-μm-tip diameter needle-electrode device. The needle electrode minimized the tissue injury when compared to a typical larger metal electrode, as confirmed by bleeding during penetration. The proposed electrode device showed both acute and chronic in vivo recording capabilities for diabetic mice while reducing the glial cells' responses. Because of these device characteristics, the 4-μm-tip diameter needle-electrode will allow electrophysiological studies on diabetes models of not only mice, as proven in this study, but also other animals.

Recent technological advancements in testing and monitoring instrumentation have greatly contributed to the progress in cancer treatment by surgical, chemotherapeutic and radiotherapeutic interventions. However, the mortality rate still remains high, calling for the development of new treatment strategies with higher efficacy. Extensive efforts driven in this direction have included broadening of early cancer screening and applying innovative theranostic nanotechnologies. They have been supported by platforms introduced to enable the detection and monitoring of cancer biomarkers, inhibitors, and other agents, able to slow down cancer progression and prevent metastasis. Despite of the well-recognized principles of the immune checkpoint blockade, the efficacy of immunotherapy achieved so far does not meet the well-founded expectations. For a successful cancer treatment, highly sensitive, robust, and inexpensive multiplex biosensors have to be designed to aid in the biomarkers monitoring and in the development of new inhibitors. In this review, we provide an overview of the efforts undertaken to aid in the development and monitoring of anticancer immunotherapy, based on the programmed cell-death immune checkpoint (PD-1/PDL-1) blockade, by designing biosensors for the detection of relevant cancer biomarkers and their inhibitors screening. This review also emphasizes alternative targets made by exosomes carrying PD-L1 overexpressed in cancer cells and passed into the excreted exosomes. Evaluated are also novel targeted drug delivery nanocarriers, providing simultaneous biosensing, thereby contributing to the emerging immune checkpoint cancer therapy. On the basis of the current trends and the emerging technologies, future perspectives of cancer diagnostics and treatment monitoring using biosensing platforms are projected.

Enzyme-free hybridization chain reaction (HCR) technology is often used as a signal amplification tool for the detection of different targets. In this study, an ultrasensitive and label-free method for detecting miRNA-21 was developed using the nanopore ionic current rectification (ICR) technology coupled with HCR technology. The probe oligonucleotide (DNA1) was combined with the gold-coated nanopore through the Au-S bond to form a DNA1-functionalized gold-coated nanopore (DNA1-Au-coated nanopore). Since miRNA-21 is partially complementary to DNA1, it can be selectively recognized by DNA1-functionalized gold-coated nanopores. The target (miRNA-21) can induce the opening of hairpin DNA and HCR reaction after the introduction of hairpin DNA H1 and H2. The concentration of miRNA-21 will affect the combination of H1 and H2 on the inner wall of the nanopore, and its surface charge will change with the internal modification, thereby changing the ion current rectification ratio. Under the condition that the concentration of H1, H2 and HCR reaction time are constant, the change of ICR ratio is linearly correlated with the logarithm of miRNA-21 concentration within a certain range, which shows that the sensing strategy we designed can achieve target miRNA-21 detection. This ultrasensitive miRNA holds great promise in the field of cancer diagnosis.

Paraquat (PQ) is a typical biotoxic small molecule. Knowledge of how to directly introduce it into cyclic amplification rather than transform it into a secondary target is lacking in current analytical methods. Considering the urgent need for trace pesticide residue detection and the inherent defects of small molecule analysis, a CRISPR/Cas12a-driven small molecule-induced dual-cycle strategy was developed based on the immune competition method. The key to signal amplification is the mutual activation and acceleration between Cycle 1 triggered by the small molecule and Cycle 2 driven by CRISPR/Cas12a. Impressively, small molecules have been successfully incorporated into the dual-cycle strategy, which achieves a low detection limit (3.1 pg/mL) and a wide linear range (from 10 pg/mL to 50 μg/mL). Moreover, the designed biosensor was successfully employed to evaluate the PQ residual level in real samples and showed effective implementation for the bioanalysis of small molecule targets and pesticide residue-related food safety.

Endoplasmic reticulum (ER) stress can induce reactive oxygen (ROS) generation which is directly associated with the emergence of atherosclerosis. Foam cells could promote atherogenesis by inducing ER stress. To understand hypochlorite (ClO-) levels in foam cells under ER stress, novel ER-targeted ClO- activatable ratiometric fluorescence probes Rx-NE and Rx-NCE were designed using a classical rhodamine dye and coumarin dye bridge moiety as the fluorescent skeleton. Both Rx-NE and Rx-NCE demonstrated ratiometric detection capabilities for ClO-, with Rx-NCE showing better sensitivity compared to Rx-NE. The probe Rx-NCE could detect the upregulation of ClO- in foam cells under ER stress and clearly outline delineation of the boundary of atherosclerotic plaques by dual-color imaging. Importantly, the hypochlorite-activated ratiometric probe Rx-NCE had been innovatively applied to the distinction of atherosclerotic blood vessels in atherosclerosis-bearing transgenic (tg) (flk1: eGFP) zebrafish. The probe Rx-NCE is of significant value for investigating the pathological role of ER stress and atherosclerotic diseases, as well as offering new insights into the identification of atherosclerosis.

Human cerebral organoids (COs), generated from stem cells, are emerging animal alternatives for understanding brain development and neurodegeneration diseases. Long-term growth of COs is currently hindered by the limitation of efficient oxygen infiltration and continuous nutrient supply, leading to general inner hypoxia and cell death at the core region of the organoids. Here, we developed a three-dimensional (3D) microfluidic platform with dynamic fluidic perturbation and oxygen supply. We demonstrated COs cultured in the 3D microfluidic system grew continuously for over 50 days without cell death at the core region. Increased cell proliferation and enhanced cell differentiation were also observed and verified with immunofluorescence staining, proteomics and metabolomics. Time-lapse proteomics from 7 consecutive acquisitions between day 4 and day 30 identified 546 proteins differently expressed accompanying COs growth, which were mainly relevant to nervous system development, in utero embryonic development, brain development and neuron migration. Our 3D microfluidic platform provides potential utility for culturing high-homogeneous human organoids.

In this work, selenium and nitrogen co-doped carbon dots (SeN-CDs) possessing highly efficient electrochemiluminescence (ECL) and excellent biocompatibility were synthesized as a new emitter with S2O82- as a coreactant for constructing a biosensor to detect microRNA-221 (miRNA-221) sensitively. Notably, the SeN-CDs exhibited superior ECL performance compared with the N-doped CDs, in which selenium with excellent redox activity served as a coreaction accelerator for facilitating the electroreduction of S2O82- to significantly improve ECL efficiency. Furthermore, target-induced T7 exonuclease (T7 Exo)-assisted double cycle amplification strategy could convert traces of target miRNA-221 into large amounts of output DNA to capture three-dimensional (3D) nanostructures (DTN-Au NPs-DOX-Fc) loaded with large amounts of ECL signal quencher. The constructed biosensor could realize ultrasensitive detection of miRNA-221 and has a low detection limit reaching 2.3 aM, with a successful application to detect miRNA-221 in lysate of Hela and MHCC97-L cancer cell. This work explored a novel method to strengthen the ECL performance of CDs to construct an ECL biosensing platform with sensitive detecting of biomarkers and disease diagnosis.

Sensitive and accurate biomarker-driven assay guidance has been widely adopted to identify responsive patients for immune checkpoint blockade (ICB) therapy to impede disease progression and extend survival. However, most current assays are invasive, requiring surgical pathology specimens and only informing monochronic information. Here, we report a multiplexed enhanced fluorescence microarray immunoassay (eFMIA) based on a nanostructured gold nanoisland substrate (AuNIS), which macroscopically amplifies near-infrared fluorescence (NIRF) of a structurally symmetric IRDye78 fluorophore by over two orders of magnitude of 202.6-fold. Aided by non-contact piezo-driven micro-dispensing (PDMD), eFMIA simultaneously and semi-quantitatively detected intracellular and secreted programmed death-ligand 1 (PD-L1) and intercellular adhesion molecule-1 (ICAM-1) in human nasopharyngeal carcinoma (NPC) cells. The assay performance was superior to fluorescence immunoassays (FIA) and enzyme-linked immunosorbent assays (ELISA), with lower detection limits. Using eFMIA, we found significantly differential levels of soluble PD-L1 (sPD-L1) and sICAM-1 in the sera of 28 cancer patients, with different clinical outcomes following anti-PD-1 ICB therapy. With a well-characterized mechanism, the high-performance plasmonic multiplexed assay with the composite biomarkers may be a valuable tool to assist clinicians with decision-making and patient stratification to afford predictive ICB therapy responses.

SARS-CoV-2 antigen detection plays a key role in the rapid diagnosis of COVID-19. However, current clinically used immunoassays are often limited by assay throughput, sensitivity, accuracy, and field operating conditions. To address these challenges, we constructed a self-enhanced electrochemiluminescence (ECL) array chip (SE2AC) for highly sensitive and label-free detection of SARS-CoV-2 nucleocapsid protein (N protein) with a facile and portable assay setup. Firstly, the self-enhanced ECL nanomaterials with inherent film-forming properties were synthesized by co-doping Ru(bpy)32+ and polyethyleneimine (PEI) in silica nanoparticles (Ru/PEI@SiO2). Secondly, a resistance-induced potential difference-based single-electrode electrochemical system (SEES) was adapted to serve as the electrode array to facilitate one-step assembly without the need for chip alignment. Thirdly, the chip electrode array was functionalized with the synthesized self-enhanced ECL emitters and captured antibodies. In addition, a portable detection box equipped with a smartphone was 3D-printed to serve as the chip holder and "dark room" for imaging acquisition. The SE2AC performance was validated with N protein with a limit of detection (LOD) of 0.47 pg/mL in the range of 1-10,000 pg/mL. Furthermore, the chip successfully detected the viral antigen residue as low as 1.92 pg/mL from diluted rehabilitation patients' serum samples. This is the first study reporting label-free detection of SARS-Cov-2 N protein based on a self-enhanced ECL immunosensor, which provides a novel facile method for highly sensitive diagnosis of COVID-19 with high throughput, portability, and low cost.

As an important emerging pollutant, antibiotic resistance genes (ARGs) monitoring is crucial to protect the ecological environment and public health, but its rapid and accurate detection is still a major challenge. In this study, a new single-labeled dual-signal output ratiometric electrochemical genosensor (E-DNA) was developed for the rapid and highly sensitive detection of ARGs using a synergistic signal amplification strategy of T3C2@Au nanoparticles (T3C2@AuNPs) and isothermal strand displacement polymerase reaction (ISDPR). Specially, two-dimensional monolayer T3C2 nanosheets loaded with uniformly gold nanoparticles were prepared and used as the sensing platform of the E-DNA sensor. Benefiting from excellent conductivity and large specific surface area of Ti3C2@AuNPs, the probe immobilization capacity of the E-DNA sensor is doubled, and electrochemical response signals of the E-DNA sensor were significantly improved. The proposed single-labeled dual-signal output ratiometric sensing strategy exhibits three to six times higher sensitivity for the sul2 gene than the single-signal sensing strategy, which significantly reduces cost meanwhile retaining the advantages of high sensitivity and reliability offered by conventional dual-labeled ratiometric sensors. Coupled with ISDPR amplification technology, the E-DNA sensor has a wider linear range from 10 fM to 10 nM and a limit of detection as low as 2.04 fM (S/N=3). More importantly, the E-DNA sensor demonstrates excellent specificity, good stability and reproducibility for target ARGs detection in real water samples. The proposed new sensing strategy provides a highly sensitive and versatile tool for the rapid and accurate quantitative analysis of various ARGs in environmental water samples.

The skin microbiome is thought to play a critical role in maintaining skin health and protecting against infection. While most microorganisms that live on the skin are harmless or even beneficial, some can cause skin infections or other health problems, emphasizing the importance of diagnosis of the composition and diversity of the skin flora. However, conventional diagnostic methods for evaluation of the skin microbiome are not sensitive enough to detect bacteria at low concentrations and suffer from poor specificity, thus limiting early diagnosis of bacterial infections. In this study, we developed novel approaches for bacterial species detection and identification methods with single-cell sensitivity using super-resolution microscopy and AI-based image analysis: a protein quantification-based method and an AI-based bacterial image analysis method. We demonstrate that these methods can differentiate between common bacterial members of the skin flora, including Staphylococcus aureus and Staphylococcus epidermidis, and different ribotypes of Cutibacterium acnes, both in purified bacterial samples and in scaling skin samples. The advantages of these methods, including the lack of time-consuming amplification or purification steps and single-cell level detection sensitivity, allow early diagnosis of bacterial infections, even from bacterial samples at extremely low concentrations, thus showing promise as a next-generation platform for microbiome detection as single-cell diagnostics.

N6-methyladenosine (m6A) is an ubiquitous post-transcriptional modification catalyzed by METTL3/14 complex in eukaryotic mRNAs. The abnormal METTL3/14 complex activity affects multiple steps of RNA metabolism and may induce various diseases. Herein, we demonstrate the RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues. METTL3/14 complex can catalyze the methylation of RNA probe to prevent it from being cleaved by MazF. The intact RNA probe is recognized by the magnetic bead (MB)-capture probe conjugates to induce duplex-specific nuclease (DSN)-assisted cyclic digestion, exposing numerous shorter ssDNAs with 3'-OH end. The shorter ssDNAs on the MB surface can act as the primers to initiate terminal deoxynucleotidyl transferase (TdT)-enhanced tyramide signal amplification (TSA), forming the Cy5 fluorescence-encoded nanostructures. After magnetic separation, the Cy5 fluorescence-encoded nanostructures are digested by DNase I to release abundant Cy5 fluorophores that can be simply quantified by fluorescence measurement. This assay achieves good specificity and high sensitivity with a detection limit of 58.8 aM, and it can screen METTL3/14 complex inhibitors and quantify METTL3/14 complex activity at the single-cell level. Furthermore, this assay can differentiate the METTL3/14 complex level in breast cancer patient tissues and healthy volunteer tissues.

At present, the 100% case fatality and the cross-infection of virus strains make the ASFV 's harm to society continue to expand. The absence of an effective commercial vaccine poses early detection remains the most effective means of curbing ASFV infection. Here, we report a cascaded detection platform based on the CRISPR-Cas12a system combined with graphene field-effect transistor sensors. The cascade platform could detect ASFV as low as 0.5 aM within 30 min and achieve typing of wild and vaccine strains of ASFV in a single detection system. The evaluation of 16 clinical samples proved that, compared with the gold standard Real-time PCR method, this platform has outstanding advantages in sensitivity, specificity and typing. Combining CRISPR-Cas12a's high specificity with the bipolar electric field effect of graphene field-effect transistor, the cascade platform is expected to achieve clinical application in the field of DNA disease detection, and provides a new direction for multi-strain disease typing.

LDs (Lipid droplets) are key organelles for lipid metabolism and storage, which are closely related to ferroptosis and fatty liver. Due to its small size and highly dynamic nature, developing high-fidelity fluorescent probes for imaging of LDs is crucial for observing the dynamic physiological processes of LDs and investigating LDs-associated diseases. Herein, we synthesized three dicyanoisophorone-based fluorescent probes (DCIMe, DCIJ, and DCIQ) with different electron-donating groups and studied their imaging performance for LDs. The results show that DCIQ is highly polarity sensitive and can perform high-fidelity imaging for LDs, with significantly better performance than DCIMe, DCIJ, and commercial LD probe BODIPY 493/503. Based on this, DCIQ was successfully applied to real-time observe the interplays between LDs and other organelles (mitochondria, lysosomes, and endoplasmic reticulum), and to image the dynamics of LDs with fast scanning mode (0.44 s/frame) and the generation of oleic acid-induced LDs with high-fidelity. Finally, DCIQ was used to study the changes of LDs in the ferroptosis process and nonalcoholic fatty liver disease tissues. Overall, this study provided a powerful tool for high-fidelity imaging of LDs in cells and tissues.

MiRNAs played critical roles in triple negative breast cancer (TNBC) as potential biomarkers. Herein, an efficient signal "off-on" mode-biosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) was successfully constructed for the miRNA-150-5p determination in TNBC. The ECL-RET regulated-sensing platform consisted of NiMn-LDHs nanoflowers, the artificially assembled phospholipid bilayers and hairpin DNA-labeled Eu-doped MoS2 QDs. Firstly, Eu-doped MoS2 QDs with high quantum efficiency were prepared as the ECL-RET donors. And NiMn-layer double hydroxides (LDHs) nanoflowers with wide UV-vis absorption spectra as the ECL-RET acceptors. Secondly, due to the hairpin DNA structure, the closed distance between ECL-RET donor-acceptor pair can quench the luminescence signal of Eu-doped MoS2 QDs. When miRNA-150-5p was captured, the hairpin DNA structure changed to a rodlike configuration and enlarged the distance between Eu-doped MoS2 QDs and NiMn-LDHs. As a result, the recovery of ECL signal can be observed as a signal "turn off-on" mode. Furthermore, the hydrophilicity of the lipid bilayer can reduce the nonspecific adsorption and improve the flexibility of the hairpin DNA efficiently. Therefore, based on the ECL-RET regulation strategy, the biosensor was employed to detect miRNA-150-5p from 10 fM to 1 nM with a detection limit of 1.5 fM. The constructed biosensor can effectively differentiate TNBC patient tumor and healthy breast fibroadenoma. The ECL-RET regulation strategy provided a new biosensing pathway for ultrasensitive detection of biomolecules and promoted the development of diagnosis and treatment of TNBC.

In this study, a polyethyleneimine (PEI)/Zr4+-functionalized nanofluidic sensing platform based on nonlinear hybridization chain reaction (NHCR) was developed for PNK activity assay. With the existence of PNK, the hairpin HPNK was cleaved by λ exonuclease, liberating the initiator T-DNA. Then T-DNA triggered the nonlinear HCR in solution and the reaction products were absorbed onto the nanopore, which changed the surface charge of nanofluidic device and could be detected by current-voltage characteristic curves. Compared to traditional linear HCR, the nonlinear HCR exhibits a higher sensitivity and order of growth kinetics, making it a powerful signal amplifier in bioanalysis. Due to the powerful amplification efficiency of nonlinear HCR, high sensitivity of the nanopore and specific recognition site of PNK/λ-Exo, an ultrasensitive and selective PNK sensing approach had been developed and applied to precisely quantitate the PNK activity with a LOD of 0.0001 U/mL. Moreover, utilizing this nanofluidic system as a foundation, we constructed a logic circuit that utilized PNK, adenosine diphosphate (ADP), and (NH4)2SO4 as input elements. ADP and (NH4)2SO4 had a crucial function in facilitating the PNK to regulate the DNA logic gate. By modifying the target and inhibitors, the nanofluidic device could detect a variety of stimuli and execute more advanced logical operations.

Static droplet array (SDA) is a pivotal tool for high-capacity screening assays, yet extraction and collection the target droplets that contain unique analytes or cells from the SDA remains one major technical bottleneck that limits its broader application. Here we present an optical-based on-demand droplet release (OODR) system by incorporating a 1064 nm laser-responsive indium tin oxide (ITO) layer into a chamber array-based droplet microfluidic chip. By focusing the 1064 nm laser onto the ITO layer, microbubbles can be created via local heating to selectively push-out the droplets from the chamber. Then the released droplet is readily exported in a one-droplet-one-tube (ODOT) manner by the inherent capillary force into pipette tip. Releasing of the droplets containing fluorescein sodium demonstrated ∼100% successful rate (9 out of 6400 droplets were successfully released) and low residual (only ∼5% of the droplet volume remains in the chamber). White or fluorescence image-based releasing of single-cell-droplets directly after cell loading or multi-cells-droplets derived from on-chip single-cell cultivation for both E. coli and yeast cells further demonstrated the wide applicability of OODR. The present system is user-friendly and has the potential to be applied in various high-throughput screening assays, including single molecule/cell analysis, drug screening, and phenotype-based cell sorting.

Bioaerosol detection technology represented by laser-induced fluorescence (LIF) cannot effectively detect bioaerosols in the presence of interferents such as plant-derived smoke, industrial waste gas, pollen and pollen debris which can produce strong non-biological fluorescence interference. To overcome this drawback, in this study, a novel method based on broad-spectrum high-efficiency magnetic enrichment and separation combined with adenosine triphosphate (ATP) bioluminescence was proposed for Escherichia coli (E. coli) bioaerosols rapid detection. First, E. coli bioaerosols mixed with interferents were collected. Core-shell Fe3O4@Polydopamine@Polyethyleneimine magnetic particles were used as bioaerosol enrichment materials to enrich E. coli bioaerosol sampling solutions. Subsequently, an ATP bioluminescence assay was performed to determine the concentration of E. coli. A linear relationship was observed between ATP bioluminescence intensity after enrichment and the E. coli bioaerosol concentration in the range of 870-49,098 particles per liter; the bioluminescence intensity measured after enrichment was significantly higher than that before enrichment, and this enrichment method provide a 6-fold better sensitivity in bioaerosol detection. More importantly, this method efficiently enriched and detected bioaerosols in plant-derived smoke. This method can effectively improve the sensitivity of ATP bioluminescence detection, and possesses the advantages of convenient operation and strong anti-interference ability. It also provides a foundation for the effective detection of bioaerosols mixed with interfering substances, and a reference for evaluating the sensitivity and anti-interference of LIF-based instruments.

Oxidative stress and excessive accumulation of the superoxide (O2.-) anion are at the genesis of many pathological conditions and the onset of several diseases. The real time monitoring of (O2.-) release is important to assess the extent of oxidative stress in these conditions. Herein, we present the design, fabrication and characterization of a robust (O2.-) biosensor using a simple and straightforward procedure involving deposition of a uniform layer of L-Cysteine on a gold wire electrode to which Cytochrome C (Cyt c) was conjugated. The immobilized layers, studied using conductive Atomic Force Microscopy (c-AFM) revealed a stable and uniformly distributed redox protein on the gold surface, visualized as conductivity and surface topographical plots. The biosensor enabled detection of (O2.-) at an applied potential of 0.15 V with a sensitivity of 42.4 nA/μM and a detection limit of 2.4 nM. Utility of the biosensor was demonstrated in measurements of real time (O2.-) release in activated human blood platelets and skeletal rat limb muscles following ischemia reperfusion injury (IRI), confirming the biosensor's stability and robustness for measurements in complex biological systems. The results demonstrate the ability of these biosensors to monitor real time release of (O2.-) and estimate the extent of oxidative injury in models that could easily be translated to human pathologies.

The global spread of environmental biological pollutants, such as antibiotic-resistant bacteria and their antibiotic resistance genes (ARGs), has emerged as a critical public health concern. It is imperative to address this pressing issue due to its potential implications for public health. Herein, a DNA paperclip probe with double-quenching function of target cyclic cleavage was proposed, and an electrochemiluminescence (ECL) biosensing platform was constructed using Ti3C2 MXene in-situ reduction growth of Au NPs (TCM-Au) as a coreactant accelerator, and applied to the sensitive detection of ARGs. Thanks to the excellent catalytic performance, large surface area and Au-S affinity of TCM-Au, the ECL performance of CdS QDs have been significantly improved. By cleverly utilizing the negative charge of the paperclip nucleic acid probe and its modification group, double-quenching of the ECL signal was achieved. This innovative approach, combined with target cyclic amplification, facilitated specific and sensitive detection of the mecA gene. This biosensing platform manifested highly selective and sensitive determination of mecA genes in the range of 10 fM to 100 nM and a low detection limit of 2.7 fM. The credible detectability and anti-interference were demonstrated in Yangtze river and Aeration tank outlet, indicating its promising application toward pollution monitoring of ARGs.

MicroRNAs (miRNAs) have emerged as important biomarkers in biomedicine and bioimaging due to their roles in various physiological and pathological processes. Real-time and in situ monitoring of dynamic fluctuation of miRNAs in living cells is crucial for understanding these processes. However, current miRNA imaging probes still have some limitations, including the lack of effective amplification methods for low abundance miRNAs bioanalysis and uncontrollable activation, leading to background signals and potential false-positive results. Therefore, researchers have been integrating activatable devices with miRNA amplification techniques to design stimuli-responsive nanoprobes for "on-demand" and precise imaging of miRNAs in living cells. In this review, we summarize recent advances of stimuli-responsive probes for the amplification-based imaging of miRNAs in living cells and discuss the future challenges and opportunities in this field, aiming to provide valuable insights for accurate disease diagnosis and monitoring.

Colorectal carcinoma (CRC) is the third most common cancer in terms of diagnosis and the second in terms of mortality. Recent studies have shown that various proteins, extracellular vesicles (i.e., exosomes), specific genetic variants, gene transcripts, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and altered epigenetic patterns, can be used to detect, and assess the prognosis of CRC. Over the last decade, a plethora of conventional methodologies (e.g., polymerase chain reaction [PCR], direct sequencing, enzyme-linked immunosorbent assay [ELISA], microarray, in situ hybridization) as well as advanced analytical methodologies (e.g., microfluidics, electrochemical biosensors, surface-enhanced Raman spectroscopy [SERS]) have been developed for analyzing genetic and epigenetic biomarkers using both optical and non-optical tools. Despite these methodologies, no gold standard detection method has yet been implemented that can analyze CRC with high specificity and sensitivity in an inexpensive, simple, and time-efficient manner. Moreover, until now, no study has critically reviewed the advantages and limitations of these methodologies. Here, an overview of the most used genetic and epigenetic biomarkers for CRC and their detection methods are discussed. Furthermore, a summary of the major biological, technical, and clinical challenges and advantages/limitations of existing techniques is also presented.

The COVID-19 pandemic shows a critical need for rapid, inexpensive, and ultrasensitive early detection methods based on biomarker analysis to reduce mortality rates by containing the spread of epidemics. This can be achieved through the electrical detection of nucleic acids at the single-molecule level. In particular, the scanning tunneling microscopic-assisted break junction (STM-BJ) method can be utilized to detect individual nucleic acid molecules with high specificity and sensitivity in liquid samples. Here, we demonstrate single-molecule electrical detection of RNA coronavirus biomarkers, including those of SARS-CoV-2 as well as those of different variants and subvariants. Our target sequences include a conserved sequence in the human coronavirus family, a conserved target specific for the SARS-CoV-2 family, and specific targets at the variant and subvariant levels. Our results demonstrate that it is possible to distinguish between different variants of the COVID-19 virus using electrical conductance signals, as recently suggested by theoretical approaches. Our results pave the way for future miniaturized single-molecule electrical biosensors that could be game changers for infectious diseases and other public health applications.

Psychological stress is a major factor contributing to health discrepancies among individuals. Sustained exposure to stress triggers signalling pathways in the brain, which leading to the release of stress hormones in the body. Cortisol, a steroid hormone, is a significant biomarker for stress management due to its responsibility in the body's reply to stress. The release of cortisol in bloodstream prepares the body for a "fight or flight" response by increasing heart rate, blood pressure, metabolism, and suppressing the immune system. Detecting cortisol in biological samples is crucial for understanding its role in stress and personalized healthcare. Traditional techniques for cortisol detection have limitations, prompting researchers to explore alternative strategies. Electrochemical sensing has emerged as a reliable method for point-of-care (POC) cortisol detection. This review focuses on the progress made in electrochemical sensors for cortisol detection, covering their design, principle, and electroanalytical methodologies. The analytical performance of these sensors is also analysed and summarized. Despite significant advancements, the development of electrochemical cortisol sensors faces challenges such as biofouling, sample preparation, sensitivity, flexibility, stability, and recognition layer performance. Therefore, the need to develop more sensitive electrodes and materials is emphasized. Finally, we discussed the potential strategies for electrode design and provides examples of sensing approaches. Moreover, the encounters of translating research into real world applications are addressed.

Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol. We show that RaMALDI imaging of various tissues effectively integrates molecular information acquired from both RSI and MALDI MSI of the same sample, which will drive discoveries in cell biology, biomedicine, and pathology, and advance tissue diagnostics.

Multiple pesticides are often used in combination to protect crops from pests. This makes rapid on-site detection of pesticide contamination challenging. Herein, we describe a method for simultaneous detection of diverse neonicotinoid pesticides using a sensor that combines neonicotinoid-specific odorant-binding protein 2 (OBP2), which was cloned from an insect chemical sensing protein and modified gold nanoparticles with local surface plasmon resonance (LSPR)-based digital nanoplasmonometry (DiNM). When neonicotinoid pesticides bind to OBP2 on gold nanoparticles, the induced LSPR shift peak wavelength is too small to be measured using conventional LSPR immunoassays. DiNM records and compares the scattered image intensity in two adjacent wavelength bands, A and B, centered on the LSPR peak. It considers both the peak shift and the relative intensity change in these two bands, resulting in a significant LSPR signal enhancement. Then the spectral-image contrast was computed as the signal response. Using this approach, we obtained excellent limits of detection (LODs) of 1.4, 1.5, and 4.5 ppb for the neonicotinoids imidacloprid, acetamiprid, and dinotefuran, respectively. Blind tests demonstrated high positive and negative rates for teas, approximately 85 and 100%, respectively. Recombinant OBP2 produced in E. coli offers several advantages over antibodies, including high yield, time savings, and cost effectiveness. Moreover, this method is highly selective and sensitive to neonicotinoids, making it practical for field use.

Designing new biochemical sensors and achieving selectivity and high-sensitivity analysis is one of main research directions for immunoassays. Herein, a liposome-amplification photoelectrochemical (PEC) immunoassay was developed using ultrathin mesoporous bismuth chloride oxide nanosheets (BiOCl MSCN) for the highly selective and sensitive detection of carcinoembryonic antigen (CEA). Based on good photocurrent response of BiOCl MSCN toward dopamine, a liposome-conjugated secondary antibody loaded with dopamine was added for specific recognition in the presence of CEA. After the lysis treatment, the liberated dopamine was injected into the three-electrode electrolytic cell to enhance the photocurrent of BiOCl MSCN. Under the optimized conditions, the constructed liposome-mediated PEC immunoassay showed high sensitivity against CEA, with a dynamic response in the linear range of 0.05 ng mL-1 to 100 ng mL-1 and a detection limit of 35 pg mL-1. The present study proposes a new approach to the liposome-mediated PEC immunoassay constructed on ultrathin mesoporous BiOCl nanosheets, which can be used to target further the study of the sensing mechanism.

The polarity and viscosity of the microenvironment are associated with the control of the onset and progression of pathological diseases, including inflammation, immuno-suppression and cancer. If appropriate treatment is neglected, alcoholic acute liver injury (AALI), the initial sign of alcoholic liver diseases, may transform into hepatic lesions. Therefore, it's crucial to create a particular probe to detect AALI swiftly and track its progression. Herein a polarity and viscosity dual-responsive crimson fluorescent probe (PPBI) was designed and developed, which can target mitochondria and lipid droplets. PPBI possesses aggregation-induced emission properties, good photostability and strong anti-interference ability against pH, metal ions, anions and biomolecules. This probe can distinguish cancer cells from normal ones using changes of green and red fluorescence, as well as identify changes in the cellular microenvironment associated with inflammatory and ferroptosis processes. In addition, changes in polarity and viscosity can be amplified by in vivo imaging in a mouse model to monitor alcohol-induced acute liver injury and to effectively detect the course of pharmacological intervention therapy. All the results suggest that PPBI could be a promising real-time fluorescence imaging tool for diagnosis and treatment of acute alcoholic liver injury.

The nonchiral sensor concept based on a sodalite (SOD) zeolite loaded CuxS (CuxS@SOD) catalyst is proposed as a sensing platform for chiral cysteine (Cys) determination. Chiral Cys is analyzed by the difference of binding capacity between CuxS catalysts. The observed current in amperometric i-t curve (A i-t C) is always positive for the L-cysteine (L-Cys), while it is negative for the D-cysteine (D-Cys). Under differential pulse voltammetry (DPV) method, the characteristic current peak for the CuxS@SOD moves to right (positive potential position) with the addition of L-Cys while it moves to left (negative potential direction) with the addition of D-Cys, respectively. Cyclic voltammetry (CV) is consistent with DPV and discusses the diffusion control mechanism. In this work, the ultra-trace determination of cysteine enantiomers reaches the limit of detection (LOD) of 0.70 fM and 0.60 fM by the highly efficient CuxS catalyst restrained in the nanosized SOD zeolite cages of the opening window pores, respectively. The sensor opens up a novel potential prospect for achiral composite in the field of chiral recognition through electrochemical methods with extra-low concentration.

Exosomes are useful for cancer diagnosis and monitoring. However, clinical samples contain impurities that complicate direct analyses of cancer-derived exosomes. Therefore, a microfluidic chip-based magnetically labeled exosome isolation system (MEIS-chip) was developed as a lab-on-a-chip platform for human epidermal growth factor receptor 2 (HER2)-positive cancer diagnosis and monitoring. Various magnetic nanoclusters (MNCs) were synthesized with different degrees of magnetization, and antibodies were introduced to capture HER2-overexpressing and common exosomes using immunoaffinity. MNC-bonded exosomes were separated into different exits according to their magnetization degrees. The MEIS-chip efficiently separated HER2-overexpressing exosomes from common exosomes that did not contain disease-related information. The simultaneous separation of HER2-and non-HER2-overexpressing exosomes provided a means of analyzing high-purity HER2-overexpressing exosomes while minimizing the contribution of non-target exosomes, reducing misdiagnosis risk. Notably, common exosomes served as a negative control for monitoring real-time changes in HER2 expression. These findings support the application of MEIS-chip for cancer diagnosis and treatment monitoring via effective exosome isolation.

Effective and convenient detection of melanoma cells with high sensitivity is essential to identify malignant melanoma in its early stage. However, the existing detection methods, such as immunohistochemical analysis, are too complicated and time-consuming to realize the convenient in vivo and in situ detection. Herein, a near infrared responsive photoelectrochemical (PEC) immunosensor is proposed with plasmonic Au nanoparticles-photonic TiO2 nanocaves (Au/TiO2 NCs) as photon harvest and conversion transducer and antibody as cell recognition unit. The micro-antibody/Au/TiO2 NCs photoelectrode can easily in vivo distinguish melanoma cells and can realize sensitive detection of melanoma cells in short time of 1 min with a lowest limit of detection of 2 cell mL-1. The PEC immunosensor strategy not only allows us to pioneeringly implement sensitive in vivo bio-detection, but also opens up a new avenue for rational design of cell recognition units and micro-electrode for universal and reliable bio-detections.

Development of specific signal reporters with signal amplification effect are highly needed for sensitive and accurate detection of pathogen. Herein, we design a colorimetric immunosensing nanosystem based on liposome encapsulated quantum dots-sized MnO2 nanozyme (MnO2QDs@Lip) as a signal reporter for ultrasensitive and fast detection of SARS-CoV-2 antigen. The pathogenic antigens captured and separated by antibody-conjugated magnetic beads (MBs) are further connected with antibody-modified MnO2QDs@Lip to form a sandwich-like immunocomplex structure. After triggered release, MnO2 QDs efficiently catalyze colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB, which can be qualitatively observed by naked eyes and quantitatively analyzed by UV-Vis spectra or smartphone platforms. By taking advantages of immuno-magnetic separation, excellent peroxidase-like catalytic activity of MnO2 QDs, and high encapsulation efficiency of MnO2QDs@Lip, ultrasensitive detection of SARS-CoV-2 antigen ranging from 0.1 pg/mL to 100 ng/mL is achieved within 20 min. The limit of detection (LOD) is calculated to be 65 fg/mL in PBS buffer. Furthermore, real clinical samples of SARS-CoV-2 antigens can be effectively identified by this immunosensing nanosystem with excellent accuracy. This proposed detection nanosystem provides a strategy for simple, rapid and ultrasensitive detection of pathogens and may shed light on the development of new POCT detection platforms for early diagnosis of pathogens and surveillance in public health.

It is difficult to show microbial growth kinetics online when they grow in complex matrices. We presented a novel strategy to address this challenge by developing a high-performance microbial growth analyzer (HPMGA), which employed a unique 32-channel capacitively coupled contactless conductivity detector as a sensing element and fixed with a CellStatz software. It was capable of online showing accurate and repeatable growth curves of well-dispersed and bad-dispersed microbes, whether they grew in homogeneous simple culture broth or heterogeneous complex matrices. Moreover, it could automatically report key growth kinetics parameters. In comparison to optical density (OD), plate counting and broth microdilution (BMD) methods, we demonstrated its practicability in five scenarios: 1) the illustration of the growth, growth rate, and acceleration curves of Escherichia coli (E. coli); 2) the antimicrobial susceptibility testing (AST) of Oxacillin against Staphylococcus aureus (S. aureus); 3) the determination of Ag nanoparticle toxicity on Providencia rettgeri (P. rettgeri); 4) the characterization of milk fermentation; and 5) the enumeration of viable pathogenic Vibrio in shrimp body. Results highlighted that the HPMGA method had the advantages of universality and effectivity. This technology would significantly facilitate the routine analysis of microbial growth in many fields (biology, medicine, clinic, life, food, environment, and ecology), paving an avenue for microbiologists to achieve research goals that have been inhibited for years due to a lack of practical analytical methods.

DNA-based enzyme-free signal amplification strategies are widely employed to detect biomarkers in low abundance. To enhance signal amplification, localized DNA reaction units which increases molecular collision probability is commonly utilized. However, the current understanding of the structure-function relationships in localized DNA signal amplification probes is limited, leading to unsatisfied performance. In this study, we introduced a coarse-grained molecular model to simulate the dynamic behavior of two DNA reaction units within a DNA enzyme-free signal amplification circuit called Localized Catalytic Hairpin Assembly (LCHA). We investigated the impact of localized distance and flexibility on reaction performance. The most efficient LCHA probe guided by simulation exhibits sensitivity 28 times greater that of free CHA, with a detection limit of miR-21 reaching 16 pM, while the least effective LCHA probe demonstrated a modest improvement of only 7 times. We successfully employed the optimized probe to differentiate cancer cells from normal cells based on their miR-21 expression levels, showcasing its quantification ability. By elucidating the mechanistic insights and structure-function relationship in our work, we aim to contribute valuable information that can save users' time and reduce costs when designing localized DNA probes. With a comprehensive understanding of how the localization affects probe performance, researchers can now make more informed and efficient decisions during the design process. This work would find broad applications of DNA nanotechnology in biosensing, biocomputing, and bionic robots.

We present a novel dual-mode DNA-walker based optical fiber biosensor (DMDW-Opt biosensor) for sensitive assay of micro-RNAs in bio-samples. In the sensor system, we develop a new strategy for the cascade amplification, DNA-walker/rolling cycle amplification (RCA), by the use of the residue track of the walker. The strategy can significantly improve the response of the sensor and avoid any tedious operation procedure. Dual-mode readouts, i.e., fluorescence and chemiluminescence, are measured independently without interfering with each other to achieve reliable and accurate analysis. Optical fibers with the surface modified by gold nanoparticles are utilized as the support for fabrication of the sensor, which would be benefit for developing miniaturized and portable sensing devices. The performance of the proposed method is evaluated by using micro-RNAs (MiR-155 and MiR-21) as the analytical target. The method is successfully applied for accurate determination of micoRNAs in human serum and MCF-7 cells. Our method can perform sensitive assays of MiR-155 with limit-of-detection as low as 97.72 fM and 11.22 fM, MiR-21 with limit-of-detection as low as 107.15 fM and 8.32 fM for the fluorescence- and the chemiluminescence-readout respectively, and the biosensor exhibits excellent specificity, reproducibility and storage stability, indicating its valuable potential applications for sensing trace-amount targets in complicated real samples.

Abusive use of β-agonists as feed additives for animals and medication is detrimental to human health and food safety. Conventional assays are restricted to a single type of β-agonists detection and cannot match the multiplexing features to perform automated, high throughput, and rapid quantitative analysis in real samples. In this research, we develop a portable automated chip system (PACS) with highly integrated automated devices in conjunction with portable microfluidic chips to provide simultaneous point-of-care testing of multiple β-agonists in the field, simplifying complex manual methods, shortening assay times, and improving sensitivity. Specifically, silicon film is used as reaction substrates for immobilizing the conjugates of β-agonists to increase the sensitivity of the assay result. Then, the PACS with a chemiluminescence imaging detector is established for automatic high-throughput and sensitive detection of Clenbuterol, Ractopamine, and Salbutamol based on the indirect immunoassay. Newly developed chip with high mixing performance can improve the sensitivity of target determination. Multiplex assays were carried out using the developed system for Clenbuterol, Ractopamine, and Salbutamol with a limit of detection of 54 pg mL-1,59 pg mL-1, and 93 pg mL-1, respectively. Except for sample preparation and coating, the detection in the PACS takes less than 47 min. A satisfactory sample recovery (86.33%-108.12%) was obtained, validating the reliability and practical applicability of this PACS. Meanwhile, the PACS enables sensitive and rapid detection of multiple β-agonists in farms or markets where lacking advanced laboratory facilities.

Severe antimicrobial resistance calls for developing rapid, sensitive and affordable methodological platform for clinical diagnosis of bacterial infection. Herein, a paper-based analytical device (PAD) for fluorescent (FL) detection and antimicrobial susceptibility test (AST) of Pseudomonas aeruginosa (P. aeruginosa) was fabricated by defining 96 hydrophilic microzones on a filter paper using a handheld stamp dipped with liquid wax. The size of microzones was designed to be consistent with traditional 96-well microplate, thus the FL signals can be collected by a commercialized microplate reader. Streptavidin was immobilized into the microzones by chitosan-glutaraldehyde crosslinking reaction, and then biotinylated bacteriophage tail fiber protein (TFP) was conjugated through biotin-streptavidin affinity system. TFP and fluorescein isothiocyanate (FITC) labeled antimicrobial peptide were used as capture agent and signal probe, respectively, for FL detection of P. aeruginosa on the PAD. The linear range for quantifying P. aeruginosa is 1.0 × 103 CFU/mL to 1.0 × 107 CFU/mL, with a detection limit of 137 CFU/mL. The PAD was also applied to conduct AST of P. aeruginosa for imipenem, meropenem, cefepime, amikacin, and gentamicin, and the results are consistent with the traditional broth dilution method. The PAD provides an affordable diagnosis platform for bacterial infection, especially in resource-limited institutes and countries.

Bottom-up neuroscience utilizes small, engineered biological neural networks to study neuronal activity in systems of reduced complexity. We present a platform that establishes up to six independent networks formed by primary rat neurons on planar complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs). We introduce an approach that allows repetitive stimulation and recording of network activity at any of the over 700 electrodes underlying a network. We demonstrate that the continuous application of a repetitive super-threshold stimulus yields a reproducible network answer within a 15 ms post-stimulus window. This response can be tracked with high spatiotemporal resolution across the whole extent of the network. Moreover, we show that the location of the stimulation plays a significant role in the networks' early response to the stimulus. By applying a stimulation pattern to all network-underlying electrodes in sequence, the sensitivity of the whole network to the stimulus can be visualized. We demonstrate that microchannels reduce the voltage stimulation threshold and induce the strongest network response. By varying the stimulation amplitude and frequency we reveal discrete network transition points. Finally, we introduce vector fields to follow stimulation-induced spike propagation pathways within the network. Overall we show that our defined neural networks on CMOS MEAs enable us to elicit highly reproducible activity patterns that can be precisely modulated by stimulation amplitude, stimulation frequency and the site of stimulation.

The development of accurate and reliable sensor for on-site detection of microcystin-LR (MC-LR), one of hazardous environmental pollutants, is highly required. Herein, a laser induced graphene (LIG)-based electrochemical aptasensor for sensitive on-site detection of MC-LR was reported. LIG electrode, the substrate of aptasensor, was prepared via thermal transfer with ethylene-vinyl acetate copolymer, and LIG acted as quasi-reference electrode to replace conventional Ag/AgCl electrode for better operability and robustness. LIG electrode provided large surface area to assemble tetrahedral DNA to absorb methylene blue (MB) for the signal amplification. For detection, the specific recognition of MC-LR with aptamer led to the stripping of tetrahedral DNA complex and further the decreased redox current of MB (IMB). Consequently, the fabricated aptasensor offered high analytical performance for MC-LR detection with a linear range of 1 × 10-2-1 × 105 pM and a detection limit of 3 × 10-3 pM, which was successfully used for water sample analysis with comparable reliability and accuracy of standard method. Furthermore, a portable detection platform by coupling of LIG-based electrochemical aptasensor with electrochemical workstation was constructed for on-site detection of MC-LR. This work offers a novel method for the on-site monitoring of MC-LR, which promotes the investigation of LIG-based electrochemical biosensing in the field of environmental analysis.

8-oxoguanine (8-oxoG) based DNA damage is the most common type of DNA damage which greatly affect gene expression. Therefore, accurate quantification of 8-oxoG based DNA damage is of high clinical significance. However, current methods for 8-oxoG detection struggle to balance convenience, low cost, and sensitivity. Herein, we have proposed and investigated the shortened crRNA mode of CRISPR-Cas12a system and greatly enhanced its signal-to-noise ratio. Taking advantages of the shortened crRNA mode, we further developed a CRISPR-enhanced structure-switching aptamer assay (CESA) for 8-oxoG. The analytical performance of CESA was thoroughly investigated via detecting free 8-oxoG and 8-oxoG on gDNA. The CESA displayed impressive sensitivity for free 8-oxoG, with detection and quantification limits of 32.3 pM and 0.107 nM. These limits modestly rose to 64.5 pM and 0.215 nM when examining 8-oxoG on gDNA. To demonstrate the clinical practicability and significance of the CESA system, we further applied it to measuring 8-oxoG levels in 7 plasma samples (Cervical carcinoma, 11.87 ± 0.69 nM VS. Healthy control, 2.66 ± 0.42 nM), 24 seminal plasma samples (Asthenospermia, 22.29 ± 7.48 nM VS. Normal sperm, 9.75 ± 3.59 nM), 10 breast-tissue gDNA samples (Breast cancer, 2.77 ± 0.63 nM/μg VS. Healthy control, 0.41 ± 0.09 nM/μg), and 24 sperm gDNA samples (Asthenospermia, 28.62 ± 4.84 VS. Normal sperm, 16.67 ± 3.31). This work not only proposes a novel design paradigm of shortened crRNA for developing CRISPR-Cas12a based biosensors but also offers a powerful tool for detecting 8-oxoG based DNA damage.

Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/μL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/μL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.

The emergence of the graphene-based hybrid electrical-electrochemical vertical device (EEVD) has introduced a promising nanostructured biosensor tailored for point-of-care applications. In this study, we present an innovative EEVD capable of simultaneously detecting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in both serum and saliva. The foundation of the EEVD lies in a poly-neutral red-graphene heterojunction, which has been enhanced with a bioconjugate of gold nanoparticles and antibodies. The biodevice demonstrates a remarkable limit of detection, registering at the femtomolar scale (2.86 fmol L-1 or 0.1 pg mL-1). Its sensitivity is characterized by a 6.1 mV/decade response, and its operational range spans 10-12 to 10-7 g mL-1 in both serum and saliva samples. With a 20.0 μL of biological samples and a rapid processing time of under 10 min, the EEVD achieves the feat of dual antigen detection. The tests achieved 100.0% specificity, accuracy, and sensitivity in saliva, and 100.0% specificity, 88.9% accuracy, and 80.0% sensitivity in serum. This study highlights the EEVD as a low-cost solution of rapid viral detection during the crucial initial phases of COVID-19 infections.

A new photoelectrochemical immunoassay based on self-assembled p-n Ag2O@Bi2O2S nanoflower heterojunction was designed and developed for quantitative monitoring of prostate-specific antigen (PSA) in biological fluids. Primarily, self-assembled p-n Ag2O@Bi2O2S nanoflower heterojunctions were served as the photoactive materials and coated onto the surface of electrodes. Subsequently, the glucose oxidase (GOx) was bound to the detection antibody (mAb2) labeled gold nanoparticles (Au NPs) and then were employed to accomplish a sandwich-like immunoreaction to generate H2O2 on a microplate incubated with monoclonal anti-PSA antibodies. In the presence of PSA, the product (H2O2) was catalyzed by the substrate, which was used as an electron sacrificial agent to improve signal conversion and capture of photogenerated electrons. Under optimum conditions, a wide linear range of 0.01-50 ng mL-1 and a low detection limit of 5.3 pg mL-1 were accomplished with the sensor, exhibiting an excellent photocurrent response. Moreover, the proposed sensor revealed satisfactory reproducibility, high selectivity, and acceptable accuracy for the real sample testing. Importantly, our work provides a novel strategy for high sensitivity detection of disease-associated biomarkers for the early diagnosis of cancers.

Breast cancer (BC) is a complex disease with high variability and no specific tumor markers available for diagnosis. Exosomes contain rich maternal tumor information and are a novel non-invasive biomarker with the potential for cancer diagnosis and prognosis. However, analysis of exosomal protein markers in blood samples is challenging due to lengthy sample workups and insufficient sensitivity. To address this difficulty, we developed a novel filter-electrochemical microfluidic chip (FEMC) to detect and classify BC directly in whole blood without requiring heavy purification methods. In our system, exosome enrichment was performed using a dual filtration system. The target was directed through a curved channel onto four screen-printed electrodes (SPEs), where it was captured by the previously modified antibodies. Simultaneously, Zr-MOFs encapsulated with a large number of methylene blue molecules (MB@UiO-66) were absorbed on the surface of exosomes due to the high affinity for phosphate groups. This process leads to the amplification of electrical signals. The approach demonstrated that the utilization of BC exosome-associated tumor biomarkers (i.e., PMSA, EGFR, CD81, and CEA), enabled the classification of various BC mouse models samples and clinical BC samples. The entire FEMC assay was completed in 1 h with a limit of detection of 1 × 104 particles/mL. Thus, the FEMC assay can provide real-time detection information, allowing timely and better-informed opportunities for clinical BC diagnosis and typing.

A smart biocatalyst should dissolve homogeneously for catalysis and recover spontaneously at the end of the reaction. In this study, we present a strategy for preparing self-precipitating enzyme catalysts by exploiting reaction-induced pH decreases, which connect the reaction extent to the catalyst aggregation state. Using poly(methacrylic acid)-functionalized gold nanoparticles as carriers, we construct smart catalysts with three model systems, including the glucose oxidase (GOx)-catalase (CAT) cascade, the alcohol dehydrogenase (ADH)-glucose dehydrogenase (GDH) cascade, and a combination of two lipases. All smart catalysts can self-separate with a nearly 100% recovery efficiency when a certain conversion threshold is reached. The threshold can be adjusted depending on the reaction demand and buffer capacity. By monitoring the optical signals caused by the dissolution/precipitation of smart catalysts, we propose a prototypic automation system that may enable unsupervised batch/fed-batch bioprocessing.

Two-photon absorbing fluorescent probes have emerged as powerful imaging tools for subcellular-level monitoring of biological substances and processes, offering advantages such as deep light penetration, minimal photodamage, low autofluorescence, and high spatial resolution. However, existing two-photon absorbing probes still face several limitations, such as small two-photon absorption cross-section, poor water solubility, low membrane permeability, and potentially high toxicity. Herein, we report three small-molecule probes, namely MSP-1arm, Lyso-2arm, and Mito-3arm, composed of a pyridinium center (electron-acceptor) and various methoxystyrene "arms" (electron-donor). These probes exhibit excellent fluorescence quantum yield and decent aqueous solubility. Leveraging the inherent intramolecular charge transfer and excitonic coupling effect, these complexes demonstrate excellent two-photon absorption in the near-infrared region. Notably, Lyso-2arm and Mito-3arm exhibit distinct targeting abilities for lysosomes and mitochondria, respectively. In two-photon microscopy experiments, Mito-3arm outperforms a commercial two-photon absorbing dye in 2D monolayer HeLa cells, delivering enhanced resolution, broader NIR light excitation window, and higher signal-to-noise ratio. Moreover, the two-photon bioimaging of 3D human forebrain organoids confirms the successful deep tissue imaging capabilities of both Lyso-2arm and Mito-3arm. Overall, this work presents a rational design strategy in developing competent two-photon-absorbing probes by varying the number of conjugated "arms" for bioimaging applications.

Conventional sensing methods report on concentrations of analytes in a single point of sampled medium or provide an average value. However, distributions of substances on surfaces of sampled objects often exhibit intricate inhomogeneities. In order to obtain snapshots of the chemical distributions on surfaces, we have developed enzyme-loaded hydrogel arrays (5 × 5 and 10 × 10). The acrylic 10 × 10 array base contains 100 holes, which are filled with agarose hydrogel containing assay enzymes and substrates. Such arrays can be exposed to the analyzed surfaces to collect minute amounts of analytes. Following a brief incubation, they are subsequently visualized in a custom-built array reader device. The reader incorporates a light-emitting diode-based light source, miniature camera, and Raspberry Pi single-board computer. Two Python programs capture and analyze the images of the array to extract pixel saturation values corresponding to individual hydrogel micropatches. The method has been thoroughly optimized for mapping of glucose and lactic acid. The optimized parameters were: contact time, agarose concentration, substrate concentration, enzyme concentration ratio, and enzyme concentration. The array biosensor was further tested by mapping glucose distribution in fruit/vegetable cross-sections (apple, guava, and cucumber) and lactic acid distribution in cheese. We think that this new hydrogel-based chemical mapping method can find applications in studies related to food science, plant physiology, clinical chemistry, and forensics; wherever the distributions of analytes on the tested surfaces need to be assessed.