Combined Molecular and Morphological Imaging of CTCs for HER2-Targeted Chemotherapy Guidance.

Since biomolecules change dynamically with tumor evolution and drug treatment, it is necessary to confirm target molecule expression in real time for effective guidance of subsequent chemotherapy treatment. However, current methods to confirm target proteins require complex processing steps and invasive tissue biopsies, limiting their clinical utility for targeted treatment monitoring. Here, CTCs, as a promising liquid biopsy source, were used to molecularly characterize the target protein HER2. To accurately identify CTCs, we specifically proposed a combined molecular and morphological imaging method, rather than using specific biomarker alone or morphology analysis, we identified CTCs as CK19+/CD45-/HE+. On the basis of the accurate identification of CTCs, we further analyzed the target protein HER2 in clinical patients at the single-CTC level. Comparative analysis of the clinical results of patient pathological tissue and paired blood samples showed that CTCs had a heterogeneous HER2 expression at the single-cell level and showed results inconsistent with the immunohistochemistry results in some cases. CTC-based analysis could help clinicians have a more comprehensive understanding of patient target protein expression. We believe that CTC-based target protein studies are of great significance for the precise management of targeted therapy.

Charge-Dependent Signal Changes for Label-Free Electrochemiluminescence Immunoassays.

Label-free electrochemiluminescence (ECL) immunoassays (lf-ECLIA), based on biomarker-induced ECL signal changes, have attracted increasing attention due to the simple, rapid, and low-cost detection of biomarkers without secondary antibodies and complicated labeling procedures. However, the interaction rule and mechanism between analytical interfaces and biomarkers have rarely been explored. Herein, the interactions between biomarkers and analytical interfaces constructed by assembly of a nanoluminophore and antibody-functionalized gold nanoparticles on an indium tin oxide electrode were studied. The nanoluminophore was synthesized by mixing Cu2+/l-cysteine chelate and N-(4-Aminobutyl)-N-ethylisoluminol-bifunctionalized gold nanoparticles with chitosan. It was found that positively charged biomarkers increased the ECL intensity, whereas negatively charged biomarkers decreased the ECL intensity. The assembly pH influenced the biomarker charges, which determined the ECL enhancement or inhibition. The detection pH only affected the ECL intensity but not the ECL changing trends. Based on the ECL signal changes, a charge-dependent lf-ECLIA was established, which exhibited inhibition responses to negatively charged human immunoglobulin G and copeptin and enhancement responses to positively charged cardiac troponin I, heart-type fatty acid binding protein, brain natriuretic peptide, and SARS-CoV-2 N protein. The linear range was 0.1-1000 pg/mL, and the detection limits were distributed in 0.024-0.091 pg/mL. Besides, a mechanism of the charge-dependent ECL enhancement and inhibition effects is proposed, which is very important for the development of new lf-ECLIA methodologies.

Sputum-Based Tumor Fluid Biopsy: Isolation and High-Throughput Single-Cell Analysis of Exfoliated Tumor Cells for Lung Cancer Diagnosis.

Timely and effective diagnosis is of great significance for improving the survival rate of lung cancer patients. Although histopathology is the main diagnostic tool among the existing methods for lung cancer diagnosis, it is not suitable for high-risk groups, early lung cancer patients, patients with advanced-stage disease, and other situations wherein tumor tissues cannot be obtained. In view of this, we proposed an innovative lung cancer diagnosis method employing for the first time a microfluidic technology for high-efficiency isolation and high-throughput single-cell analysis of exfoliated tumor cells (ETCs) in sputum. This method fully combines the advantages of traditional sputum cytology and microfluidic technology and realizes the diagnosis of lung cancer by using a small amount of repeatable ETCs instead of the tumor tissue. This method is expected to provide a practical strategy for the non-invasive detection of lung cancer patients and lung cancer screening for high-risk groups.

Potential-Resolved Differential Electrochemiluminescence Immunosensor for Cardiac Troponin I Based on MOF-5-Wrapped CdS Quantum Dot Nanoluminophores.

Recently, nanoluminophores with the potential-resolved multicolor electrochemiluminescence (PRMCECL) property have emerged and shown promising applications in sensitive, selective, and accurate bioassays, bioimaging, and multicolor emitting devices. However, only limited PRMCECL nanoluminophores and their applications in ratiometric biosensors eliminating proportional errors have been reported. Herein, a novel PRMCECL nanoluminophore was synthesized by encapsulating CdS quantum dots (CdSQDs) into MOF-5 (CdSQDs@MOF-5). Using K2S2O8 as a coreactant, two electrochemiluminescence (ECL) peaks, ECL-1 centered at 685 nm and ECL-2 centered at 475 nm, were observed at -1.4 and -1.8 V, respectively. Related ECL mechanisms have been proposed. Based on the potential-resolved ECL signals, a label-free differential ECL immunosensor for the determination of cardiac troponin I (cTnI) was established by assembly of poly(diallyldimethylammonium chloride), CdSQDs@MOF-5, and cTnI antibody-functionalized silver nanoparticles on the surface of the fluorine-doped tin oxide electrode subsequently. In the presence of cTnI, cTnI was captured by the sensing interface, leading to an increase in ECL-1 and ECL-2 intensity. cTnI could be determined in the range of 0.01-1000 pg/mL with a detection limit of 5.01 fg/mL using the intensity difference between ECL-1 and ECL-2. This work provides a new family member of PRMCECL nanoluminophores. The proposed label-free differential ECL immunosensor provides a new strategy based on potential-resolved ECL signals, which could effectively eliminate the additive error and show better sensitivity, selectivity, and accuracy for the detection of cTnI than the single-signal strategy and ratiometric strategy.

Potential-Resolved Multicolor Electrochemiluminescence of N-(4-Aminobutyl)-N-ethylisoluminol/tetra(4-carboxyphenyl)porphyrin/TiO2 Nanoluminophores.

Most electrochemiluminescence (ECL) studies involve single luminophore with a unique emission process, which severely limits its applications. Recently, multicolor ECL has attracted considerable interests. Herein, we report a novel nanoluminophore prepared by coating 5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrin (TCPP) and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) on the surface of TiO2 nanoparticles (TiO2-TCPP-ABEI), which exhibited unique potential-resolved multicolor ECL emissions using H2O2 and K2S2O8 as coreactants in an aqueous solution. Three ECL peaks, ECL-1 at 458 nm, ECL-2 at 686 nm, and ECL-3 at 529 nm, were obtained with peak potentials of 1.05, -1.65, and -1.85 V, which were attributed to the ECL emission of ABEI, TCPP, and TiO2 moiety of the nanoluminophores, respectively. Potential-resolved multicolor ECL from a nanoluminophore was observed for the first time in an aqueous solution. It opens a new research area of multicolor ECL of nanoluminophores, which is of great importance in ECL field from fundamental studies to practical applications.

Biomimetic synergistic effect of redox site and Lewis acid for construction of efficient artificial enzyme.

In enzymatic catalysis, the redox site and Lewis acid are the two main roles played by metal to assist amino acids. However, the reported enzyme mimics only focus on the redox-active metal as redox site, while the redox-inert metal as Lewis acid has, to the best of our knowledge, not been studied, presenting a bottleneck of enzyme mimics construction. Based on this, a series of highly efficient MxV2O5·nH2O peroxidase mimics with vanadium as redox site and alkaline-earth metal ion (M2+) as Lewis acid are reported. Experimental results and theoretical calculations indicate the peroxidase-mimicking activity of MxV2O5·nH2O show a periodic change with the Lewis acidity (ion potential) of M2+, revealing the mechanism of redox-inert M2+ regulating electron transfer of V-O through non-covalent polarization and thus promoting H2O2 adsorbate dissociation. The biomimetic synergetic effect of redox site and Lewis acid is expected to provide an inspiration for design of enzyme mimics.

Three-Biomarker Joint Strategy for Early and Accurate Diagnosis of Acute Myocardial Infarction via a Multiplex Electrochemiluminescence Immunoarray Coupled with Robust Machine Learning.

Acute myocardial infarction (AMI) represents a leading cause of death globally. Key to AMI recovery is timely diagnosis and initiation of treatment, ideally within 3 h of symptom onset. Cardiac troponin T (cTnT) is the gold standard yet a low cTnT result cannot rule out AMI at early times. Here, we develop a three-biomarker joint strategy for early and accurate diagnosis of AMI via an electrochemiluminescence (ECL) immunoarray coupled with robust machine learning. The ECL immunoarray is based on an array microchip with a single-electrode and chemiluminescent immuno-Gold (ciGold) nanoassemblies. The ciGold immunoarray was obtained by successively assembling nanocomposites of Cu2+/cysteine complexes and N-(aminobutyl)-N-(ethylisoluminol) bifunctionalized gold nanoparticles combined with chitosan and antibody conjugated gold nanoparticles on the surface of a microchip. Three biomarkers, including cardiac troponin I, heart type fatty acid binding protein, and copeptin, were simultaneously detected in 260 serum samples from patients presenting with chest pain by an innovative multiplexed ECL immunoarray, and classified via the three-biomarker joint assessment model using support vector machines. The model achieved perfect discrimination (100% sensitivity and specificity) for AMI vs non-AMI patients, substantially higher than cTnT alone. Within 12 h of symptom onset, high-sensitivity cardiac troponin T (hs-cTnT) misclassified >20% of patients, while the joint biomarker assessment model retained perfect accuracy. As the time between symptom onset and testing became shorter, the degree to which the joint assessment model outperformed hs-cTnT increased. The proposed three-biomarker joint strategy is obviously superior to hs-cTnT for early and accurate diagnosis of AMI, hopefully reducing AMI mortality and saving limited medical resources.