Alzheimer's disease (AD) is a common neurodegenerative disorder. Compared to the limited specificity of single-biomarker detection, the combined detection of multiple biomarkers can significantly improve the accuracy of AD diagnosis. In this study, a field-effect transistor (FET) biosensor array integrating four independently functionalized regions was fabricated by modifying carbon nanotube (CNT) surfaces with amyloid bovine serum albumin (AL-BSA) nanofilm as an antifouling layer, enabling the simultaneous detection of multiple tau proteins. The proposed FET biosensor exhibited excellent anti-fouling performance while maintaining high sensitivity, with detection limits as low as 3.3 fg/mL, 0.17 fg/mL, 0.02 fg/mL, and 2.5 fg/mL for p-tau181, p-tau217, p-tau231, and t-tau, respectively. Evaluation of 35 clinical serum samples indicated that p-tau181 outperformed the other three tau proteins as an AD diagnostic biomarker. By further incorporating a support vector machine (SVM)-based machine learning algorithm and employing multi-biomarker co-detection, the accuracy of AD diagnosis can be improved to nearly 100%. This study provides a novel strategy for array FET biosensors with high clinical application potential for the precise detection of AD.

The development of reagent-free biosensors that can continuously monitor proteins in complex biological fluids is still a critical unmet need. Molecular pendulum sensors have recently emerged as a promising reagent-free platform, enabling direct biomolecule detection without interference from surrounding matrices. However, conventional molecular pendulums employ only a single recognition element, which constrains their tunability and regulatory range. To overcome this limitation, a molecular pendulum design incorporating dual recognition elements was introduced here. By simultaneously engaging two nonoverlapping epitopes on human serum albumin (HSA) or Aβ42 monomers, the dual recognition pendulum underwent a marked conformational re-orientation that amplified the intrinsic signal gain. Replacing one aptamer with a high-affinity antibody further increased target binding efficiency, boosting charge transfer efficiency and delivering substantially larger electrical read-out than the corresponding dual-aptamer architecture. Besides, cryogenic surface modification promoted rapid self-assembly of the probes, further lowering the detection limit. This design also enabled rapid regeneration and reuse of the sensor by allowing reset completion within 30 s through adjustment of applied voltage and time. The sensor demonstrated excellent specificity even in the presence of multiple interferents and exhibited high sensitivity in biological samples (serum and artificial cerebrospinal fluid).

Lung cancer is a leading cause of cancer-related mortality globally, with complex biochemical processes underlying its initiation and progression. Nitroxyl (HNO), a reactive nitrogen species with unique chemical and biological properties, has emerged as a potential regulator of tumor biology. However, the transient nature and intertwined pathways of HNO with nitric oxide (NO) pose significant challenges for precise, real-time detection at the subcellular level, particularly within the mitochondrial key sites where HNO is generated and utilized. Herein, a mitochondria-targeted fluorescent probe (DCPH) was designed and synthesized for the super-resolution imaging of HNO. DCPH demonstrates high sensitivity and selectivity for HNO, with stable performance across the physiological pH range. Super-resolution imaging confirms the mitochondria-targeting capability of DCPH, which enables the visualization of both exogenous and endogenous HNO in A549 cells. This probe is capable of successfully monitoring HNO dynamics under inflammatory stimulation and during NO/H2S crosstalk. Furthermore, in vivo imaging in tumor-bearing mice demonstrates that DCPH can visually track HNO production during sodium nitroprusside (SNP) treatment, which is correlated with significant tumor growth inhibition. Overall, DCPH provides a novel tool for the high-resolution detection of mitochondrial HNO and offers insights into the biological functions of HNO in cancer.

Nanoscale distribution of the mesenchymal-toepithelial transition factor (Met) on the cell surface is essential for intracellular signaling and cell behavior. Met oligomerization activates cell behavior, and ligand spacing is crucial to this activation mechanism. However, the optimal spatial distance for Met oligomerization-mediated activation remains unknown, hindering the design of related cellular function modulators. Herein, based on the programmability of aptamers, beta-cyclodextrin (beta-CD)-mediated DNA nano-modulators with different ligand spacings were designed to dynamically modulate the lateral distance between receptors on cell membranes, studying the effects of ligand distances on Met oligomerization activation. Four hybridization chain reaction (HCR)-based DNA nano-modulators (S-HCR18/28/44/60) with 18-60 bp spacings were self-assembled via complementary base pairing. The results showed that DNA nano-modulators with 28-and 44-bp spacings were most effective in activating Met. This regulation was also designed to be reversible via competitive displacement of beta-CD. For example, reversible Met activation was achieved using adamantane (Ada) to displace S-HCR28 and S-HCR44. Additionally, the four DNA nano-modulators had varying efficacy in repairing acetaminopheninduced liver injury, with S-HCR28 and S-HCR44 being more effective. This work could guide the design of efficient cellular receptor modulators and offer spatial distance insights for cell biology.

Oral cancer is a prevalent malignancy in the head and neck region. The development of non-invasive and efficient early diagnostic methods is crucial for improving cure rates and the quality of life for patients. Here, we developed a portable microfluidic detection platform combining magnetic force and electrowetting force for simultaneous detection of two tumor biomarkers, carcinoembryonic antigen (CEA) and matrix metalloproteinase-1 (MMP-1) in saliva. The detection platform integrated a portable droplet manipulation device (digital microfluidic) and a smartphone-based signal acquisition device. During the detection process, targets were firstly captured from saliva samples using antibody-conjugated magnetic beads (MBs), then achieving target separation and enrichment. Subsequently, the enriched target-bound MBs and other reagents were loaded into the device. Driven by the magnet, the MBs sequentially accomplished the steps of capturing gold nanoparticles (AuNPs), followed by washing, releasing of barcode DNA from AuNPs. Next, under the control of electrowetting force, the droplet containing barcode DNA was driven to mix with the fluorescent substrate droplet, triggering CRISPR-Cas autocatalytic fluorescence signal amplification. Finally, the fluorescence signal was acquired using a smartphone. The platform achieves detection limits of 0.01ng/mL for CEA and 0.03ng/mL for MMP-1, which meet the clinical testing requirements. This digital microfluidic platform is easy to operate, and low-cost, offering an efficient tool for the early diagnosis of oral cancer.

Exosomes, rich in diverse bioactive molecules, have emerged as a promising "liquid biopsy" approach for early cancer screening. However, relying solely on a single biomarker type can easily lead to missed diagnoses or misjudgments. Therefore, there is an urgent need to develop multiple biomarkers detection strategies to facilitate precise diagnosis. Herein, a microfluidic chip integrating two signals (ALP-driven chemiluminescence and nanobeacons-based fluorescence) into a single device was developed using breast cancer exosomes as a model. A simple sample injection enabled capture and quantitative detection of exosomal surface proteins (CD63, EpCAM, HER2) and internal miRNAs (miRNA-21, miRNA-375). After capture, protein levels were determined by driving magnetic beads and capturing images with a smartphone. Prefabricated nanobeacons were then directly introduced into the chip, enabling miRNA signal detection without RNA extraction. This five-marker biomarker panel achieved a diagnostic accuracy of 99.6% in breast cancer identification, representing an improvement of at least 4.6% over single-marker approaches and an 8.6% increase over any individual biomarker. This multitarget exosome analysis platform may provide a highly sensitive, low-cost, and easily scalable novel tool for breast cancer early diagnosis and treatment efficacy assessment.

Ammonia nitrogen is one of the key indicators in water quality monitoring, and its rapid, efficient on-site detection is critical for timely water pollution control. Herein, a gravity-driven microfluidic chip with pre-stored reagents was developed for the detection of ammonia nitrogen in surface water. Specifically, 10 mg of ammonia nitrogen solid reagents were pre-stored within the detection chamber of chip. The reagents, consisting of an oxidant, a chromogenic agent, a catalyst, and multiple auxiliary agents, exhibited long-term stability under the conditions of 25 degrees C away from light in N2, with only 2.9% signal degradation within one year. Furthermore, the gravity-driven chip eliminated the need for an external pump and simplified operation. During on-site detection, users only needed to add 200 mu L of water sample into the sample chamber, then the water spontaneously flowed through the detection chamber under gravity, rapidly dissolving the reagents and initiating chromogenic reaction. The entire process could be completed within 15 min, and a color signal was acquired using a smartphone, thereby eliminating the need for specialized instruments. This method exhibited a linear relationship within the concentration range of 0.1-1 mg/L with a detection limit of 0.09 mg/L (S/N = 3), which is below the regulatory limits for ammonia nitrogen in both Chinese Class I standards for surface water and the European Directive for drinking water. With its ease of operation, low reagent consumption, and long shelf life, the chip can serve as a powerful tool for rapid screening of ammonia nitrogen in surface water.

The merits of an ideal wearable sensor are high sensitivity, excellent stability, and user-friendly operation, typically requiring room-temperature storage and sample-to-answer detection capability. Here, we developed aptamer-functionalized multivalent fluorescent DNA nanostructures (Ap-MFDNs) and demonstrated that they can improve the detection sensitivity for sweat biomarkers, i.e., cortisol, lactate, and uric acid, compared with monovalent probes. The results showed that the multivalent binding strategy exhibited varying effects on enhancing the detection sensitivity for different small molecules, which was particularly evident for cortisol. Molecular docking was employed to help understand these differences. Meanwhile, we demonstrated that the Ap-MFDNs can be stored as a lyophilized powder under appropriate ionic strength. Based on the multivalent DNA nanostructures, we designed corresponding wearable sensors for direct detection of the above three targets in human sweat with the aid of smartphones. The detection range of the wearable sensor can cover the physiological levels of the three small molecules in sweat. Therefore, multivalent DNA nanostructures have the potential to improve the performance of wearable sensors.

The pathogenesis of Alzheimer's disease (AD) was complex, including excessive deposition of β-amyloid (Aβ), microglia dysfunction, and neuroinflammation. Therefore, single-pathway treatment was not sufficient to ameliorate the multifaceted pathological changes associated with AD. Moreover, the low permeability of blood-brain barrier (BBB) and the lack of AD locus selectivity further limited the intervention efficacy of current AD drugs. In this study, a novel nanoparticle coating was designed by hybridizing the membrane from brain microvascular endothelial cell exosomes and macrophage exosomes, and combining polydopamine nanoparticles, resveratrol and Aβ-targeting aptamers to construct engineered exosomes (RPDA@Rb-A) with multiple targeting capabilities to intervene in Aβ clearance and regulate microglial dysfunction. Based on the homing effect of brain microvascular endothelial cell exosomes and the natural inflammation targeting ability of macrophage exosomes, RPDA@Rb-A can easily penetrate the blood brain barrier and accumulate in the brain inflammation site after capturing Aβ aggregates. RPDA@Rb-A can effectively intervene in AD through multi-pathway, including degraded toxic Aβ aggregates through local heating induced by near-infrared laser irradiation and alleviated neurotoxicity, promoted microglial clearance of Aβ by capturing Aβ, and modulated microglia-induced neuroinflammation by efficient delivery of small molecule drugs. In AD mouse model, the administration of RPDA@Rb-A resulted in a significant reduction in amyloid plaque deposition, neuroinflammation, and cognitive impairments. The engineered exosomes based on membrane hybridization overcome the shortcomings of traditional drug carriers in poor penetration and insufficient targeting to the central nervous system, and provide a potential platform for multi pathways intervention in AD.

The high sensitivity of field-effect transistor (FET) biosensors has made them a valuable tool for detecting low abundance biomarkers in AD diagnosis, but it faces ongoing challenges, particularly in their susceptibility to interference from complex sample matrices. Here, an amyloid-like nanofilm was introduced as an intermediate layer to enhance the antifouling ability and sensitivity of FET biosensors in complex systems. This nanofilm serves a dual purpose: due to the size-selective mechanism of the amyloid-like nanofilm, which prevents interference from nonspecific proteins, the proposed biosensor exhibited enhanced stability and antifouling capability in complex samples (nonspecific response of less than 5%). Weakening Debye shielding through its undulating porous structure enabled the highly sensitive detection of biomarkers even in solutions with high ionic strength. The biosensor successfully detected Alzheimer's disease (AD) biomarker P-tau181 with a low limit of detection down to 0.1 fg/mL and achieved a remarkable 100% diagnostic accuracy across 25 serum samples. This study provides a highly stable and sensitive FET biosensor that is expected to be used for early screening of AD.

Aptamers make up a class of nucleic acid strands that can specifically recognize target molecules. However, the selection of aptamers typically involves multiple optimizations and an iterative process, including library design, high-affinity library enrichment, sequencing, and affinity assessment of candidate sequences. Among these steps, the affinity evaluation of large numbers of candidate sequences is particularly resource-intensive in terms of labor, materials, and time. To address this, we developed a low-cost digital microfluidic platform based on printed circuit boards using the aptamer of small-molecule tumor drug mertansine as an example. We achieved miniaturization and automation of the affinity determination process. First, we optimized the droplet actuation parameters on the digital microfluidic platform and verified its feasibility for aptamer affinity determination. Then, using this platform, we investigated the affinity of seven candidate sequences for mertansine and successfully found two new aptamers (Seq15-X and Seq19-X) with good affinity for mertansine. The dissociation constants (Kd) of the two aptamers are 70.9 ± 8.5 nM and 43.6 ± 21.6 nM, respectively. Compared with manual methods, the digital microfluidic platform not only reduced the time for a single determination from 105 to 45 min but also significantly decreased the reagent consumption from 1280 to 30.72 μL. Therefore, the digital microfluidic platform offers several advantages, including high automation, low reagent consumption, and strong versatility, not only significantly improving the efficiency of aptamer screening and performance evaluation but also providing a promising tool for the study of interactions between other biomolecules.

Cell receptors are key regulators of cellular signaling. However, on-demand reversible engineering of cell receptors to intervene in cellular behavior remains a challenge. Herein, a reversible receptor engineer strategy (SL1/NCDP/Ada) was developed. Initially, ferrocene (Fc)-modified aptamer (SL1-Fc) engaged in biorecognition with the mesenchymal epidermal transition factor (Met) receptor. Subsequently, β-cyclodextrin polymers (β-CDPs) recognized SL1-Fc through host-guest interactions, spatially engineering the Met receptor and, consequently, influencing cell proliferation and migration behavior (SL1/CDP strategy). In addition, due to the stronger host-guest recognition of adamantane (Ada) and β-CD, Ada could compete with SL1-Fc to bind CDPs, causing CDPs to be released from the cell surface, thereby eliminating the regulatory effect of SL1/CDPs and achieving reversible regulation of cell migration behavior. This approach employed cross-networked (NCDP) and linear cyclodextrin polymers (LCDP). The results showed that SL1/NCDP could induce Met receptor aggregation and activated the Met receptor due to the compact network structure of NCDP. In contrast, SL1/LCDP could not induce Met receptor aggregation due to the large spatial spacing of monomers in LCDP. However, SL1/LCDP could significantly inhibit ligand hepatocyte growth factor (HGF)-induced Met receptor activation by targeting the Met receptor and indirectly regulate cell proliferation and migration behavior. This work provides a novel strategy to reversibly engineer cell receptors in a customized manner to regulate cell proliferation and migration behavior on demand.

The challenge of developing sensing platforms for the direct monitoring of targets within complex samples is well-recognized. To address this, a one-step electrochemical sensing detection platform was introduced, featuring an innovative Y-shaped DNA molecular pendulum design. The approach deviated from the conventional molecular pendulum mode by employing a split aptamer instead of a full one, thereby enabling the detection of small molecules and low-molecular-weight proteins. Three Y-shaped DNA molecular pendulum configurations were designed: the single-arm, the flexible double-arm, and the stable double-arm Y-shaped DNA molecular pendulum. The results revealed that the Y-shaped scaffold pendulum with a stable two-armed structure not only offered a broader detection range for target concentrations but also produced a more substantial electrical signal enhancement compared to other modes. This enhanced performance is attributed to the stable conformation of this design, which prolongs the time the probe takes to overcome fluid resistance and reach the electrode surface, leading to a more significant alteration in the electrical signal. The sensor can be utilized for one-step detection of enrofloxacin (ENR) in diluted samples (milk, artificial urine, and cosmetics), and its detection range (0.001-100 ng/mL) is fully compliant with the EU maximum residue levels (100 ng/mL) for ENR in milk. Additionally, the sensor can detect myoglobin (Myo) in artificial urine and serum by simply changing the recognized DNA strand. This work provided a simple, expandable idea for the detection of small molecules and low-molecular-weight proteins.

Drug-induced liver injury (DILI) has emerged as an urgent clinical challenge. It is characterized by mitochondrial dysfunction in liver cells, which leads to abnormal changes in H2O2 levels within the mitochondria. Super-resolution imaging allows for the observation of the fine structure of mitochondria at the nanometer scale, potentially enabling the detection of mitochondrial H2O2 levels during DILI at the subcellular organelle level. Here, we report the design and synthesis of a novel H2O2-activated probe for the detection of mitochondrial H2O2 levels. SML is a near-infrared (NIR) fluorescent probe with a large Stokes shift (260 nm) and a sensing mechanism based on intramolecular charge transfer (ICT) switching. Super-resolution imaging of mitochondrial H2O2 was conducted using structured illumination microscopy (SIM). The improved accuracy in observing periods of mitochondrial dysfunction allows the SML probe to be effectively utilized for the rapid monitoring nanoscale upregulation of H2O2 during DILI and hepatic fibrosis, thus providing SML with the capability to screen for effective therapeutic candidates.

The isolation and detection of exosomes as tumor markers are of vital importance for the early diagnosis, therapeutic monitoring, and mechanistic studies of tumors. Here, exosomes derived from breast cancer cells were chosen as model targets, and a wash-free, enzyme-free, handheld mini centrifugation method based on hydrogels was developed to effectively isolate and detect breast cancer exosomes. Dual aptamers (CD63-T1 and EpCAM-T2) were employed to specifically recognize and capture breast cancer exosomes. This specific recognition triggered the formation of hybridization chain reaction (HCR) nanostructures on the captured exosomes through the interaction of hairpin 1 and the alginate complex (H1-Alg) and hairpin 2 (H2-Cy3). The interaction of Ca2+ and alginate enabled the in situ formation of a hydrogel on the exosome surface. Subsequent low-speed centrifugation using a handheld mini centrifuge facilitated the efficient isolation of the exosomes, thereby eliminating the need for tedious washing steps. Utilizing the classical chelation reaction of ethylene diamine tetraacetic acid (EDTA) with Ca2+, the hydrogel can be rapidly cleaved for enzyme-free release of exosomes. The method demonstrated excellent capture and release efficiencies of approximately 85% and 98%, respectively, for specific cancerous exosomes. Notably, the exosomes isolated by the hydrogel system retained excellent biological activity, making them suitable for further analysis and potential applications. Meanwhile, the highly sensitive detection of breast cancer exosomes based on this strategy could also be achieved with a lower limit of detection as low as 3.2 × 103 particles/mL. This work provides a novel and cost-effective strategy for the effective isolation and detection of tumor-derived exosomes, which can help to promote the subsequent application of exosomes in research.

To facilitate on-site detection by nonspecialists, there is a demand for the development of portable "sample-to-answer" devices capable of executing all procedures in an automated or easy-to-operate manner. Here, we developed an automated detection device that integrated a magnetofluidic manipulation system and a signal acquisition system. Both systems were controllable via a smartphone. In the device, the mixing of solutions and magnetic beads in the static chamber was enhanced by steel bead agitation, which improved the reaction efficiency. We demonstrate the performance of the device using myoglobin detection as an example. During the detection process, the plasma was separated from the whole blood sample using a homemade mini-centrifuge, and subsequently, the plasma, magnetic beads, and reagents were added to a magnetofluidic chip with multiple chambers. After the chip was loaded, the device was initiated with a smartphone App via Bluetooth. Then, the magnetic beads were shuttled through different chambers of the chip and multiple steps were completed automatically: first, the targets were separated and enriched using antibody-modified magnetic beads, followed by washing, binding with aptamer-functionalized G-quadruplex, signal amplifying (optional), and chromogenic reaction. Finally, the images of colored solutions were captured and processed by a smartphone to obtain the concentrations of myoglobin. The detection limits depended on the mode of signal conversion, which were 0.1 or 2.7 nM (with or without signal amplifying). With its simple operation, compact design, low cost, and ease of scalability, this automated detection device holds potential applications in human health, food safety, environmental monitoring, etc.

Detection of breast cancer-derived exosomes can help provide valuable information for the diagnosis of breast cancer. However, accurate detection of breast cancer exosomes remains challenging. Herein, taking SK-BR-3 exosome, a HER2-positive breast cancer exosome, as target model, a dual-mode assay based on complementary scattering signals for accurate detection of breast cancer-originated exosomes was constructed by proximity ligation mediated RCA reaction. To assure accuracy, three probes recognition (consisting of EpCAM, HER2, and MUC1 aptamers for the recognition of proteins on the surface of exosomes), proximity ligation reaction, and complementary scattering signals produced by the gold nanoparticles (Au NPs) in the supernatant and the magnetic beads-Au NPs (MBs-Au NPs) in the precipitate were used. This assay was equipped to distinguish HER2positive breast cancer exosomes from HER2-negative and normal exosomes. Due to the RCA reaction triggered by proximity ligation, the dual-mode assay can detect SK-BR-3 exosome with a concentration of 1.0 x 102 particles/ mu L. With excellent accuracy and sensitivity, the assay can also distinguish healthy individuals from breast cancer patients, making it suitable for clinical application. This work was expected to provide an effective tool for accurate detection of breast cancer-derived exosomes.

Wearable sweat sensors have achieved rapid development since they hold great potential in personalized health monitoring. However, a typical difficulty in practical processes is the control of working conditions for biorecognition elements, e.g., pH level and ionic strength in sweat may decrease the affinity between analytes and recognition elements. Here, we developed a wearable sensing device for cortisol detection in sweat using an aptamer as the recognition element. The device integrated functions of sweat collection, reagent prestorage, and signal conversion. Especially, the components of prestored reagents were optimized according to the inherent characteristics of sweat samples and electrodes, which allowed us to keep optimal conditions for aptamers. The sweat samples were transferred from the inlet of the device to the reagent prestored chamber, and the dry preserved reagents were rehydrated with sweat and then arrived at the aptamer-modified electrodes. Sweat samples of volunteers were analyzed by the wearable sensing device, and the results showed a good correlation with those of the ELISA kit. We believe that this convenient and reliable wearable sensing device has significant potential in self-health monitoring.

High-affinity, specific, and sensitive probes are crucial for the specific recognition and identification of tumor cells from complex matrices. Multivalent binding is a powerful strategy, but the irrational spatial distribution of the functional moieties may reduce the probe performance. Here, we constructed a Janus DNA triangular prism nanostructure (3Zy1-JTP-3) for sensitive detection and specific isolation of tumor cells. Benefiting from spatial features of the triangular prism, the fluorescence intensity induced by 3Zy1-JTP-3 was almost 4 times that of the monovalent structure. Moreover, the DNA triangular prisms were connected to form hand-in-hand multivalent DNA triangular prism structures (Zy1-MTP), in which the fluorescence intensity and affinity were increased to 9-fold and 10-fold of 3Zy1-JTP-3, respectively. Furthermore, 3Zy1-JTP-3 and Zy1-MTP were combined with magnetic beads, and the latter showed higher capture efficiency (>90 %) in whole blood. This work provides a new strategy for the efficient capture of rare cells in complex biological samples.

Exosomes generated from tumors contain a range of bioactive molecules, making them attractive and trustworthy non-invasive biomarkers for prognostic monitoring and adjuvant diagnosis of malignancies. Here, a homogeneous portable electrochemical sensing platform based on DNAzyme-induced DNA walker and enzymecatalyzed amplification strategies has been created to accurately identify exosomes from breast cancer. The aptasensor induced enzymatic cleavage through dual probe recognition, and the continuous movement of the DNA walker achieves the cleavage of a large number of substrate strands. This causes a significant number of enzyme -containing DNA fragments to be released, and the enzyme that is released catalyzes the production of hydrogen peroxide (H2O2) by oxidizing excess glucose. The hydrogen peroxide (H2O2) underwent a redox reaction using a screen -printed carbon electrode (SPCE), and the detection of exosome can be achieved by reading current signal changes. The detection limit of the sensor is 3.63 x 104 particles/mL SK -BR -3 exosomes. This method improved the accuracy of detection by double recognition of MUC1 protein and HER2 protein on breast cancer exosomes and avoided cumbersome electrode modification steps. In addition, clinical serum samples can be analyzed using the constructed electrochemical sensor to separate patients with breast cancer from healthy volunteers as well as patients with various tumors. It is anticipated that the current work will give an effective tool for the accurate detection of breast cancer exosomes because it presents a novel technique for doing so in HER2-positive breast cancer.