Here, we present a modular and programmable real-time quantitative detection platform that integrates ligase chain reaction (LCR) with multicomponent nucleic acid enzymes (MNAzymes) for single-nucleotide variant (SNV) analysis. By incorporating a universal MNAzyme-based reporter, the LCR-MNAzyme system enables PCR-like real-time quantitative monitoring of SNVs while supporting programmable and multiplex detection. The target recognition module allows precise quantification of multiple variants, while reconfiguring the signal output module enables multiplex detection in a single homogeneous reaction. To further enhance analytical performance, glyoxal-caged probes were incorporated to achieve hot-start LCR, effectively suppressing background amplification and improving detection sensitivity. The optimized system exhibits a linear range from 10 aM to 10 pM with a detection limit of 5.79 aM and enables quantification of the JAK2 V617F mutation down to a variant allele frequency of 0.03%. Quantitative results from clinical samples showed excellent agreement with sequencing data. This work establishes a generalizable framework for highly sensitive, specific, and multiplex SNV detection, advancing the development of quantitative nucleic acid analysis tools for precision molecular research.

Hemin-loaded amphiphilic nanoparticles (HeNPs) were engineered to stabilize hemin and enhance its catalytic performance in the luminol-H2O2chemiluminescence (CL) system. The confined hydrophobic microenvironment effectively suppressed hemin aggregation and produced a stable glow-type emission, which could be directly captured by a smartphone without external excitation or optical accessories. By integrating this stabilized chemiluminescent platform with an acetylcholinesterase (AChE) inhibition mechanism, organophosphorus pesticides (OPs) were translated into quantifiable CL responses. The smartphone-based readout enabled sensitive and reliable analysis with good linearity. The proposed method was further validated in lake water and Chinese cabbage samples, where the results obtained by smartphone imaging showed good agreement with those from microplate-based CL detection and GC-MS. Overall, this work demonstrates a smartphone-enabled chemiluminescent sensing strategy for monitoring OP residues in food and environmental samples.

The sensitive detection of proteins with a simple colorimetric readout is of great significance for the early diagnosis of diseases and the monitoring of therapeutic efficacy. Herein, we report a novel colorimetric immunoassay that integrates the dopamine-mediated signal amplification with Cell Counting Kit-8 (CCK-8) detection. Through the construction of a sandwich immunoassay system, the horseradish peroxidase conjugated to the detection antibody rapidly catalyzes the deposition and in situ self-polymerization of dopamine on the immune-complex, forming a versatile nanoscale polydopamine (PDA). The resulting PDA serves as a universal scaffold for high-density binding of lactic dehydrogenase (LDH). Upon the addition of L-lactic acid and nicotinamide adenine dinucleotide (NAD+), the bound LDH efficiently catalyzes the conversion of L-lactic acid to pyruvate, and simultaneously reduces NAD+ to NADH, which subsequently drives the colorimetric reaction of CCK-8 system, producing a strong absorbance signal at 450 nm. This process greatly enhances the immunoassay sensitivity by converting immune recognition events into a CCK-8 colorimetric readout and integrating multi-step cascade signal amplification. This method exhibits a broad dynamic range spanning five orders of magnitude for the model analytes, carcinoma embryonic antigen and alpha-fetoprotein, with detection limit as low as 5.4 pg/mL and 4.1 pg/mL respectively. Moreover, the distinct color gradient enables reliable semi-quantitative assessment with the naked eye. This colorimetric immunoassay provides a general, straightforward, and potent method in clinical diagnostics and prognosis of diseases.

In the detection of RNA biomarkers, conventional PCR-based workflows such as reverse transcription-PCR (RT-PCR) and ligation-PCR each exhibit inherent limitations. RT-PCR requires cDNA synthesis and often struggles with targets of high sequence similarity or atypical length and structure. Ligation-PCR improves sequence discrimination by enzymatically joining adjacent probes only when perfectly matched, yet conventional two-step formats-where ligation and amplification are performed separately-introduce workflow complexity, increased handling, and contamination risk. To overcome these challenges, we developed a glyoxal-assisted one-pot ligation-PCR assay that integrates probe ligation and PCR amplification within a single closed-tube system. The method employs thermally responsive glyoxal-caged primers that remain inactive during the ligation phase and are gradually activated during PCR cycling, thereby preventing premature extension and minimizing nonspecific amplification. Validated primarily on mRNA splice variants, the assay achieved sensitivity comparable to conventional two-step ligation-PCR while providing markedly improved discrimination among closely related splice isoforms. Additional experiments demonstrate the feasibility of extending this strategy to microRNA detection. This streamlined one-tube strategy simplifies operation, reduces contamination risk, and establishes a robust and efficient one-pot ligation-PCR framework that is readily adaptable to different RNA targets for precise RNA biomarker detection.

Organophosphorus (OPs) pesticide residues pose significant threats to human health and the environment. To tackle this issue, we synthesized water-soluble fluorescent conjugated polymer nanoparticles (WSCPNs), which offer high fluorescence intensity, simple preparation methods, and ease of functionalization, making them ideal candidates for fluorescent sensing applications. These WSCPNs were subsequently used to prepare a WSCPNs@MnO2 probe via in situ synthesis, resulting in efficient fluorescence resonance energy transfer between WSCPNs and MnO2. This system effectively oxidizes non-fluorescent o-Phenylenediamine (OPD) into 2,3-diaminophenazine (DAP). In the absence of OPs, acetylthiocholine (ATCh) is catalyzed by acetylcholinesterase (AChE) to produce thiocholine (TCh), which reduces MnO2 on the surface of the probe, restoring the fluorescence intensity. When OPs are present, AChE's catalytic pathway is inhibited, limiting the recovery of fluorescence intensity in WSCPNs. The remaining MnO2 can further oxidize OPD to DAP, allowing quantitative analysis by monitoring changes in fluorescence signal ratios, achieving a detection limit of 0.0139 ng/mL. Additionally, color changes can be captured and analyzed using a smartphone, facilitating fluorescence visualization for OPs detection, achieving a detection limit of 0.025 ng/mL. This method exhibits excellent anti-interference capabilities and has been successfully applied to detect organophosphorus pesticides in leaves and soil, demonstrating the effectiveness of our ratiometric fluorescence and fluorescence visualization dual-mode sensing platform for monitoring OPs.

Ultrasensitive quantification of low-abundance proteins is critical for disease full-cycle management but remains a significant challenge. Herein, we present an immunotriggered double stem-loop probe-mediated isothermal amplification (IDSP-IA) assay for highly sensitive and specific protein detection. The IDSP-IA assay consists of three modular components: a monoclonal capture antibody (mAb1)-coated reaction tube for target capture, an aptamer-oligonucleotide switch (Apt-OS) for signal transduction, and a universal double stem-loop-mediated isothermal amplification module. After target protein capture and enrichment of mAb1-coated tubes, Apt-OS converts nonamplifiable protein signals to the nucleic acid triggers, initiating efficient exponential amplification. Benefiting from the modular design, the assay allows flexible adaptation to various proteins by simply replacing antibody/aptamer pairs while keeping amplification components unchanged. We have demonstrated that the IDSP-IA assay achieves a wide dynamic range (5 fg/mL to 50 pg/mL) with a limit of detection (LOD) of 1.3 fg/mL for α-fetoprotein (AFP) and 1.4 fg/mL for interleukin-6 (IL-6) detection without cross-reactivity from other common serum proteins. More importantly, clinical validation using human serum samples showed excellent correlation with routinely used electrochemiluminescence and chemiluminescence immunoassays. These advantages, including high specificity, ultrasensitivity, modular design, and universality, enable the IDSP-IA assay to provide a robust and flexible tool for accurate and attomolar protein biomarker quantification in diagnostic and biomedical settings.

The development of simple and ultrasensitive immunoassays is critical for the early diagnosis and treatment of diseases. The efficient integration of amplification strategies with highly sensitive detection probes plays a key role in boosting the ultrasensitive immunoassays. In this paper, we report a multifunctional and integrated dopamine-modified conjugated polymer nanoparticle (DA-CPN) probe prepared by a one-step nanoprecipitation method for ultrasensitive immunoassay. The multifunctional DA-CPNs fully integrate the unique properties of dopamine and fluorescent conjugated polymer nanoparticles, while possessing three key capabilities. 1, DA-CPNs can be rapidly deposited onto adjacent proteins catalysed by horseradish peroxidase (HRP) labelled in the detection antibody in a sandwich immunoassay. 2, DA-CPNs undergo self-polymerization simultaneously, resulting in the assembly of large numbers of CPNs and thereby amplifying the detection signal. 3, CPNs possess excellent fluorescence brightness and strong photobleaching resistance, further enhancing the sensitivity of fluorescence immunoassays while simplifying the experimental procedure. Using carcinoma embryonic antigen (CEA) as a model, the proposed method demonstrated a wide linear range spanning five orders of magnitude and an exceptional sensitivity with a detection limit of 0.39 fg/mL. Therefore, this study based on DA-CPNs provides a versatile and highly promising platform for the ultrasensitive immunoassays and in vitro diagnosis of diseases.

In this assay, based on the terminal protection of small-molecule-linked DNA, a new ultrasensitive real-time fluorescence strategy combined with an isothermal exponential amplification reaction (IEXPAR) has been established for protein assay. By the clever design of DNA, terminal protection is combined with efficient IEXPAR. The target protein explicitly binds to small molecules attached to the template DNA, protecting the template DNA from exonuclease I (Exo I) degradation. The added DNA primer hybridizes with the protected template DNA and triggers the following IEXPAR. IEXPAR has a super amplification efficiency of 10(6)-10(9) times. The IEXPAR yields numerous double-stranded DNA (dsDNA) molecules. The fluorescence dye SYBR Green I (SG), which is sensitive to dsDNA, is used to determine the real-time fluorescence of the IEXPAR. Conversely, without the target protein, the template DNA is hydrolyzed by Exo I, failing to trigger the IEXPAR. The intriguing combination of IEXPAR and terminal protection realizes the ultrasensitive detection of protein. As low as 100 fmol L-1 SA and 200 pg mL(-1) folic acid (FR) are accurately detected.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.

The development of simple, sensitive, and efficient methods for antibiotic detection is of great significance for human health and environmental protection. Currently, although the competitive immunoassays are popular for detecting antibiotic molecules, they need to label antibiotic or specific antibody with signaling molecule. The development of noncompetitive immunoassay for antibiotic detection has always faced significant challenges. Herein, we have developed a light-assisted noncompetitive immunoassay for the detection of antibiotics based on their photosensitive characteristic. Firstly, the antibody coated on the microplate specifically captures the target antibiotic. Afterward, under light irradiation, the captured photosensitive antibiotic produces reactive oxygen species (ROS), which oxidizes the added ROS dye to generate a fluorescent signal. Therefore, a noncompetitive immunoassay for antibiotic detection is achieved by utilizing the unique property of photosensitive antibiotics to generate ROS instead of labeling signaling molecules. By using antibiotics demeclocycline hydrochloride (DMCO) and polymyxin B (PMB) as research models, the proposed noncompetitive fluorescence immunoassay can specifically detect as low as 5 ng/mL DMCO and 70 ng/mL PMB, respectively. The light-assisted noncompetitive immunoassay strategy opens a new avenue for simple, sensitive, label-free, and high-throughput detection of photosensitive antibiotics or photosensitive drugs. Based on the unique photosensitive property of antibiotics, a light-assisted noncompetitive immunoassay is developed for the detection of photosensitive antibiotics by integrating a one-step immunoassay and the fluorescence detection of reactive oxygen species under light irradiation. The light-assisted noncompetitive immunoassay strategy opens a new avenue for the simple, sensitive, label-free, and high-throughput detection of photosensitive antibiotics or photosensitive drugs.image

The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.

We report a photosensitive polymyxin B-modified conjugated oligomer nanoparticle that integrates the targeted identification and synergistic photodynamic therapy in one treatment against resistant Gram-negative bacteria. The study expands the application of antibiotics and opens a new avenue for enhancing photodynamic antimicrobial therapy and fighting bacterial resistance.

Hydrazine is a kind of widely used industrial raw material and a toxic biochemical reagent. Due to its toxic to organisms, hydrazine has been classified to be a hazardous environmental pollutant. It is urgent to develop fluorescent probe tools for selective sensitivity detection of hydrazine in the environment and the body. We developed here a new coumarin-based fluorescent probe for hydrazine detection. The probe can selectively detect hydrazine over other environmental and endogenous interfering analytes with a large off-on fluorescence response. The detection limit is 8.55 ppb, which is well below the allowed threshold limit value. The sensing mechanism is hydrazine-induced pyrazole ring formation, which is confirmed by HRMS and DFT calculation methods. Additionally, the probe could also be applied for hydrazine imaging in living HeLa cells.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10−4 to 5 U/mL with four orders of magnitude, and the detection limit is 2.7 × 10−4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

G-quadruplex/Hemin (G4/Hemin) complex has been widely used in biocatalysis and analytical applications. Meanwhile, compared with natural proteinous enzyme, its low catalytic activity is still limiting its applications. Even though several methods have been developed to enhance the peroxidation efficiency, the important core of the G4 design based enhancement mechanism is still indistinct. Here, we focus the mechanism study on the two most important microdomains: the iron porphyrin center and the catalytic synergy group within the 3′ flanking. These microdomains not only provide the pocket for the combination of substrate, but also offer the axial coordination for the accelerated formation of Compound I (catalytic intermediate). In order to obtain a more suitable space layout to further accelerate the catalytic process, we have used the bases within the 3′ flanking to precisely regulate the distance between microdomains. Finally, the position-dependent effect on catalytic enhancement is observed. When dC is positioned at the second-position of 3′ flanking, the newly obtained DNAzyme achieves an order of magnitude improvement compared to parent G4/Hemin in catalytic activity. The results highlight the influence of the distance between the catalytic synergy group and iron porphyrin center on the activity of DNAzyme, and provide insightful information for the design of highly active DNAzymes.

The irrational or excessive use of antibiotics causes the emergence of bacterial resistance, making antibiotics less effective or ineffective. As the number of resistant antibiotics increases, it is crucial to develop new strategies and innovative approaches to potentiate the efficacy of existing antibiotics. In this paper, we report that some existing antibiotics can produce reactive oxygen species (ROS) directly under light irradiation. Thus, a novel antibacterial photodynamic therapy (PDT) strategy is proposed by using existing antibiotics for which the activities are potentiated via light-activation. This antibiotic-based PDT strategy can achieve efficient bacteria killing with a low dosage of antibiotics, indicating that bacterial killing can be enhanced by the light-irradiated antibiotics. Moreover, the specific types of ROS produced by different antibiotics under light irradiation were studied for better elucidation of the antibacterial mechanism. The findings can extend the application of existing antibiotics and provide a promising strategy for treatment of bacterial infections and even cancers.

A novel and multifunctional oligonucleotide-functionalized conjugated oligomer nanoparticle (CON) is developed for effective cancer cell imaging and therapy by integration of fluorescence imaging, target recognition, photodynamic therapy (PDT), and chemotherapy. The CON itself possesses the high fluorescence emission efficiency for imaging and can generate reactive oxygen species for PDT. Due to its excellent biocompatibility, easy functional modification, and efficient drug loading and release ability, oligonucleotides enable the CON to be functionalized in many forms. For the target recognition, the CONs are functionalized with oligonucleotides labeled with folate molecules, which can specifically bind the CONs to the tumor cells overexpressing folate receptors. Moreover, the designed oligonucleotide on the CON hybridizes with its complementary sequence to form double-stranded DNA, which enables a sequence-specific loading with doxorubicin for chemotherapy. For different situations, the therapy of chemotherapy and PDT can be used solely or combinatorially. Therefore, this study provides a facile strategy for the multifunctional modification of CONs with oligonucleotides and opens a new avenue for targeted cell imaging and tumor treatment.

基于水溶性氟硼二吡咯类共轭聚合物PBF可与长波长区(＞600 nm)荧光染料产生荧光共振能量转移(FRET),结合引物延伸反应,发展了一种均相检测miRNA的新方法.方法首先通过引物延伸反应在目标miRNA分子上引入荧光染料Cy5,形成miRNA-Cy5.然后,PBF与miRNA-Cy5/DNA杂合体通过静电力结合并发生从PBF到Cy5的有效FRET,实现了基于阳离子共轭聚合物(CCP)的长波长区miRNA的均相检测,方法灵敏度高、特异性好,测定miR-221的线性为15～6 000 pmol/L,检出限(3σ)为8.4 pmol/L.方法拓展了CCP的应用,为基于CCP的生物传感和生化分析提供了新的均相检测平台.

Sensitive detection of uracil-DNA glycosylase (UDG) activity is very important in the study of many fundamental biochemical processes and clinical applications. Here, we develop a novel assay for the detection of UDG activity by using the self-initiating multiple rolling circle amplification (SM-RCA) strategy. We first design a trigger probe modified with NH2 at its 3'-terminal and uracil base in the middle of sequence, which is complementary to a cyclized padlock probe. In the presence of UDG, a uracil base can be excised by UDG to generate an apurinic/apyrimidinic (AP) site. The AP site is recognized and cleaved by endonuclease IV (Endo IV), releasing the primer with 3'-OH. The primer can trigger the rolling circle amplification (RCA) reaction, producing a long and repeated DNA strand embedded some uracil bases. These uracil bases can be cleaved by UDG and Endo IV again, and then, more primers are generated to initiate SM-RCA reaction, producing large amounts of DNA product. Afterward, the DNA product is measured by a specific DNA fluorescence dye for quantitative detection of UDG activity. The linear range of the method is 5 × 10-5 to 1.25 × 10-3 U/mL, and the detection limit is 1.7 × 10-5 U/mL. This method not only utilizes the target UDG itself to trigger RCA but also further induces SM-RCA reaction, providing a simple, sensitive, and cost-effective strategy for the detection of glycosylase and clinical diagnosis.

Nitroxyl (HNO) plays an important role in multiple physiological and pathological processes, but the detailed generation mechanism of the endogenous HNO still remained to explore and perfect further. There is an urgent need to develop an excellent fluorescent probe for selective recognition and sensitive detection of HNO in biological systems. Near-infrared (NIR) fluorescent probes with a large Stokes shift are an ideal tool for bioimaging applications. Here, we have developed a NIR fluorescent probe with a large Stokes shift, namely, NIR-HNO, to monitor HNO in cells and zebrafish. NIR-HNO consists of an isophorone-fused NIR fluorescence reporter and a diphenylphosphinobenzoyl HNO-responsive unit. Based on an aza-ylide intramolecular ester aminolysis reaction, NIR-HNO showed a rapid selective NIR fluorescent turn-on response for HNO, high sensitivity (detection limit was 39.6 nM), and large Stokes shift (265 nm). The biological imaging results indicate that NIR-HNO is a good candidate for imaging of endogenous HNO in living systems.