Honglan Qi

Ultrasensitive detection of protein biomarkers at lower concentrations is critical but remains a challenge. Here, an artificial intelligence (AI)-enhanced electrogenerated chemiluminescence (ECL) imaging method was developed for single-molecule detection. Imaging single biomolecules is realized by confining and amplifying the signal from single-molecule reaction events on magnetic beads (MBs), with amplification via TSA using a self-designed and synthesized tyramide-conjugated ruthenium complex. Resolving individual signals from single-molecule reaction events is achieved by a dual deep-learning-assisted image processing method (named as ATT-PIX, by coupling the ATTBeadNet model and PIX2PIX model) combined with an adaptive threshold method. Such an AI-enhanced ECL imaging method facilitates the spatial deconvolution of single MBs from aggregated MBs and supports robust quantitative analysis at the single-molecule level. Additionally, 91.78% F1 for "Reference field" images and 91.75% AUC (area under the curve of the receiver operating characteristic curve) for ECL images were obtained in this process. Leveraging Poisson distribution, AI-enhanced ECL imaging, and TSA, a digital intelligence ECLIA was proposed for ultrasensitive detection of the protein biomarker IL-6 (a clinical biomarker of acute inflammatory diseases) with a limit of detection (LOD) of 50 fg/mL (2.4 fM), which is better than using conventional automated ECLIA (LOD of 33 pg/mL). This work demonstrates that integrating AI with ECL imaging paves the way for next-generation digital platforms for single-molecule detection.

Catalytic activities of single-atom catalysts (SACs) are strongly correlated to metal center valence states. Herein, we report a simple and facile electrogenerated chemiluminescence (ECL) method for the sensitive and quantitative assessment of iridium (Ir) valence states in UiO-67-supported SACs, using luminol as an ECL luminophore. Three UiO-67-Ir SACs (UiO-67-Ir-1/2/3) with tunable Ir valence states were synthesized by varying the solvent types during preparation. Atomic dispersion of Ir on UiO-67 was confirmed via atomic-resolution high-angle annular dark-field scanning transmission electron microscopy, with Ir anchored through Ir(III)-nitrogen coordination. To elucidate the influence of Ir valence states on the ECL behavior, we systematically investigated the catalytic effects of the three UiO-67-Ir catalysts on both cathodic (luminol-O₂) and anodic (luminol-H₂O₂) ECL systems. Different Ir valence states induced distinct types and amounts of reactive oxygen species (ROS), governing ECL responses. Compared to Ir(III), Ir(0) enhanced ROS generation, promoting both cathodic and anodic ECL intensities of luminol. Notably, the ECL intensity exhibits a positive correlation with the relative content of Ir(0): the higher the Ir(0) ratio, the greater the ECL signal. Furthermore, a linear relationship was established between the amount of Ir(0) and the ECL intensity, enabling the quantitative determination of the Ir(0) content in the SACs. This ROS-mediated ECL method offers a novel, sensitive, and quantitative alternative for assessing metal valence states in SACs, which is crucial for the rational design and optimization of high-performance SACs for catalytic applications.

The development of a sensitive and accurate method for the determination of cardiac troponin I (cTnI), as an ideal biomarker of acute myocardial infarction (AMI), is critical for the early diagnosis of AMI. In this work, a tetrahedral DNA framework (TDF)-bridged aptasensing method was proposed for the ultrasensitive determination of cTnI via electrogenerated chemiluminescence (ECL) and fluorescence imaging methods, in which a specific anti-cTnI aptamer-tagged tetrahedral DNA framework (TDF-aptamer) was designed as a capture probe by incorporating a specific aptamer against cTnI within the three-dimensional TDF. The TDF-aptamer was modified onto a gold electrode surface through the Au–S bond, providing a specifically captured platform for cTnI with a 3D binding space at the nanoscale. One microsized signal probe (Ab2-biotin•SA@MB-Ru1) was synthesized by using magnetic Fe3O4 as carriers to load a large amount of ruthenium complex and a specific cTnI antibody. After the sandwich assay among the modified TDF-aptamer, cTnI, and the signal probe, both ECL and fluorescence signals were recorded for the cTnI assay with desirable selectivity and high sensitivity (linear range of 5 to 80 pg/mL using ECL intensity detection and 0.5 to 500 pg/mL using ECL and fluorescence imaging methods). This work demonstrated that the combination of the specific TDF-aptamer and microsized Ab2-biotin•SA@MB-Ru1 within the imaging method can enhance the specific recognition and detection sensitivity for cTnI, which is promising for AMI diagnosis.

This work reports one Ru(bpy) ₃ ²⁺ ‐N,N‐dibutylethanolamine (DBAE) coreactant‐sodium dodecyl sulfate (SDS) enhancer system on an automated electrochemiluminescence immunoassay (ECLIA) analyzer for the ECL immunoassay of carbohydrate antigen 19‐9 (CA19‐9). The ECL intensity of the MB@SA•biotin‐GSK‐Ru1 bio‐conjugates in DBAE solution on the automated ECLIA analyzer, which employs a platinum working electrode under the “Heterogeneous” model, demonstrates a 333% enhancement in comparison with that of the conventional tri‐n‐propylamine coreactant. Subsequently, the developed novel DBAE coreactant‐based automated ECL method was estimated by the determination of CA19‐9, while the MB@SA•biotin‐Ab1 was taken as the capture probe and the Ru(bpy) ₃ ²⁺ derivative‐labeled CA19‐9 antibody (Ab2‐Ru1) was employed as signal probe. In the presence of the SDS enhancer, the relative ECL intensity of the formed “sandwich” MB@SA•biotin‐Ab1/CA19‐9/Ab2‐Ru1 biocomplexes exhibits a 4.3‐fold enhancement on the automated ECLIA analyzer. Notably, under the optimized conditions, the increased ECL intensity was directly in proportion to the concentration of CA19‐9 in the range from 0.5 to 8.0 U/mL with a limit of detection of 0.25 U/mL. This approach has the potential to be extended to magnetic bead‐based automated ECL methods for the sensitive detection of clinical protein biomarkers.

The development of a sensitive method for in situ monitoring of hydrogen peroxide (H₂O₂) released from living cells is crucial for disease diagnosis. In this work, we have fabricated a gold-coated optical fiber electrode (GOFE) for in situ monitoring of H₂O₂ released from living cells via transmitted electrogenerated chemiluminescence (ECL). The cylinder GOFE was fabricated by sputtering a conductive Au layer (∼100 nm) on the surface of the cylinder and the cross-section (one proximal end) of the plastic optical fiber, which was 2.4 cm in length and 2.0 mm in diameter. The prepared GOFE was further modified with luminol-conjugated gold nanoparticles (Luminol-AuNPs) on the proximal end as a transmitted sensing platform (abbreviated as Luminol-AuNPs/GOFE). Its proximal end was taken as the venue for sensing and ECL generation, while its distal end was taken as the readout spot for the ECL emission. Two detection modes, including transmission mode and nontransmission mode, were developed for ECL detection. The transmitted ECL intensity at the distal end via transmission mode was directly proportional to the concentration of H₂O₂ in the range from 50 μM to 1.0 mM with a limit of detection of 12.5 μM. Moreover, the developed Luminol-AuNPs/GOFE-based transmitted ECL method was successfully employed to monitor the release of H₂O₂ from living A549 cells in situ under lipopolysaccharide stimulation. This work presents a promising method for fabricating optical electrodes with favorable electroactive and transmittance properties for in situ and sensitive monitoring of biomolecules in different analytical applications.

Electrochemiluminescence immunoassay (ECLIA), recognized for its good sensitivity and selectivity, has been widely commercialized in the field of in vitro diagnostics. Despite its advancements, the determination of protein biomarkers at concentrations below pg/mL remains a significant challenge in automated ECLIA. Here, a new magnetic rolling circle amplification-assisted automated ECLIA (ECLIA-RCA) method was proposed for ultrasensitive determination of protein biomarkers. The presence of protein biomarkers could specifically drive the formation of magnetic immunocomplexes between the magnetic bead (MB)-based capture antibody, the target protein, and the single-stranded DNA-tagged secondary antibody. Subsequent triggering of the RCA reaction and signal labeling enables the hybridization of a substantial number of Ru(bpy)₃²⁺ derivative-labeled single-stranded DNA to the elongated DNA linkers attached to each magnetic immunocomplex, thereby significantly amplifying the ECL signal. By integrating RCA with MB separation, the developed ECLIA-RCA method realized sensitive determination of cardiac troponin I (cTnI) with a detection limit of 0.1 pg/mL. The method also demonstrates excellent selectivity with minimal interference from nontarget proteins. Validation using 19 clinical human serum samples revealed a good linear correlation of the measured cTnI concentrations between the ECLIA-RCA results and clinical results (R² = 0.9471). Additionally, the versatility of this automated ECLIA-RCA approach was successfully demonstrated through the quantification of alpha-fetoprotein, achieving a detection limit of 0.3 pg/mL. The ECLIA-RCA method holds great promise for the ultrasensitive determination of protein biomarkers, offering significant potential for early disease diagnosis.

Simultaneous detection of dopamine (DA) and serotonin (5-HT) provides useful information in the early diagnosis of neurological diseases. However, existing approaches have to be conducted in centralized laboratories with assay complexity. Herein, we developed a bifunctional fluorescence aptasensor based on target-induced strand displacement, which enables the simultaneous detection of DA and 5-HT in multiple body fluids. Cy5/FAM-tagged aptamers were first hybridized with a Dabcyl-quenched complementary strand ss-DNA1-(Dabcyl)₂ to form a low fluorescence background complex Cy5-ds-DNA-(Dabcyl)₂-FAM. Upon target binding (DA/5-HT), the tagged aptamers were selectively displaced, restoring Cy5 (red) and FAM (green) fluorescence upon excitation with 600 and 465 nm, respectively. The aptasensor exhibits high sensitivity, with a limit of detection of 0.3 nM for DA and 0.5 nM for 5-HT in solution. Good recovery in artificial cerebrospinal fluid and urine and results consistent with those of high-performance liquid chromatography (HPLC) were achieved. With the success in solution-based detection, we further transferred the aptasensor onto glass fiber paper (GFP) to develop a paper-based aptasensor. Good recovery in tears and urine and results matching those of HPLC were also obtained. This simple and bifunctional fluorescence aptasensor offers a promising point-of-care platform for the detection of multiple neurotransmitters in diverse body fluids.

The global incidence of flash floods has increased, leading to fatalities and significant economic losses. Hence, the development of cost-effective, timely, and efficient flood early warning systems holds significant economic and social value. In this work, a flash flood alarm device was fabricated that employed water-responsive xerogel as a core material and utilized smartphone electrogenerated chemiluminescence (ECL) imaging as the detecting technique. The water-responsive ECL xerogel was derived from the drying process of a conductive ECL hydrogel, where the conductive ECL hydrogel material was prepared by doping both Ru(bpy)₃Cl₂ luminophore and conductive indium tin oxide nanoparticles into the flexible hydrophilic polymer matrix via the crosslinking of poly(vinyl alcohol) with borax and sucrose. Intriguingly, the ECL xerogel exhibits a rapid response to water and displays a significantly enhanced ECL behavior in the presence of water. Moreover, one flood alarm device was fabricated by the incorporation of one serrate FTO electrode as the anode, five pieces of the ECL xerogel, a strip-type FTO electrode as the cathode, a battery group as the power supply, and a smartphone as the detection device. This flood alarm device demonstrates effective performance in water level warning functionalities even in dim or nocturnal conditions with cost-effectiveness, good sensitivity, and resolution, which are associated with the greatest risk of flooding. This work provides a flood alarm device based on an ECL xerogel utilizing smartphones as detectors, which is promising for the early warning of flood.

A signal-on electrochemiluminescence (ECL) immunoassay was developed for the detection of the SARS-CoV-2 N protein using coreactant-encapsulated polymersomes as tags and a graphitic carbon nitride (g-C₃N₄) nanosheet-modified screen-printed carbon electrode (SPCE) as the working electrode. Poly(ether imide) (PEI)-modified g-C₃N₄ nanosheets were prepared through ultrasonic exfoliation followed by a supramolecular covalent scaffold strategy. Spray coating, a scalable deposition technique, was employed to modify the SPCE surface with g-C₃N₄ nanosheets. After thermal curing, the nanosheets adhered firmly to the electrode via binders in the carbon ink, yielding a stable modified SPCE. Immunosensors were constructed by immobilizing capture antibodies on the g-C₃N₄-modified SPCE. Potassium persulfate, (K₂S₂O₈), the coreactant of g-C₃N₄, was encapsulated in polymersomes to prepare ECL tags, and probes were fabricated by conjugating SARS-CoV-2 N protein binding aptamers to these polymersomes. Upon recognition of the target protein and sandwich complexes formed on the immunosensor, the subsequent addition of Triton X-100 disrupted the polymersomes, releasing large amounts of K₂S₂O₈, which markedly enhanced the ECL mission of g-C₃N₄ nanosheets. This amplification enabled highly sensitive detection of the SARS-CoV-2 N protein. These findings demonstrate that controlled release of encapsulated coreactants from polymersome tags is an effective strategy to trigger g-C₃N₄-based ECL emission for sensitive viral protein detection.

Electrogenerated chemiluminescence (ECL) methods have been widely used in clinical diagnosis. Although ECL peptide-based biosensors continue to grow with good sensitivity and signal flexibility, little emphasis has been placed on the effect of the peptide sequence on ECL sensitivity. We herein studied the nuanced effects of different peptide sequences on the analytical performance of ECL peptide-based biosensors for matrix metalloproteinase 2 (MMP-2) assay, in which [(pbz)₂Ir(DMSO)Cl] (pbz = 3-(2-pyridyl)benzoic acid) was used as the ECL emitter while a specific peptide was used as the molecular recognition element. [(pbz)₂Ir(DMSO)Cl] was labeled onto the peptide by a coordination-based site-specific labeling strategy to form the ECL probe. It was found that the incorporation of one histidine (H) or two H at the terminal of the peptide resulted in a significant increase in ECL signal and a negligible effect on the cleavage ability of the ECL probe while the incorporation of H at the middle of the peptide caused a slight increase of ECL signal and a significant inhibitory effect on the cleavage ability of the ECL probe. One type of ECL peptide-based biosensor was fabricated for the determination of MMP-2 at gold nanoparticle-modified glassy carbon electrode. MMP-2 can be detected with a detection limit of 0.9 ng/mL, and it can be applied to detect MMP-2 in serum samples and cell secretions. This work provides some information for the synthesis of efficient ECL probe and for the design of ECL peptide-based biosensors, which are promising in clinical diagnosis.