Bispecific Nanobody-Aptamer Conjugates for Enhanced Cancer Therapy in Solid Tumors.

Bispecific antibodies possess exceptional potential as therapeutic agents due to their capacity to bind to two different antigens simultaneously. However, challenges pertain to unsatisfactory stability, manufacturing complexity, and limited tumor penetration hinder their broad applicability. In this study, a versatile technology is presented for the rapid generation of bispecific nanobody-aptamer conjugates with efficient tumor penetration. The approach utilizes microbial transglutaminase (MTGase) and click chemistry to achieve site-specific conjugation of nanobodies and aptamers, which are termed nanotamers. The nanotamers recognize and bind to two types of molecular targets expressed on cancer cells. As a prototype, a bispecific nanotamer is developed that binds both clusters of differentiation 47 (CD47) and mesenchymal epithelial transition receptor (Met) expressed on the tumor cell membrane. This CD47-Met nanotamer demonstrates high affinity and specificity toward tumor cells expressing both targets, exhibits improved receptor functional inhibition through a strong steric hindrance effect. Moreover, its capacity for deep tumor penetration greatly enhances the impact of conventional chemotherapy on antitumor efficacy. The as-developed nanotamer synthesis approach shows promise to customize bispecific molecular probes targeting different cancer types and different therapeutic goals.

Formaldehyde Cross-Linking-Assisted Phase Separation for Protein Aptamer Selection.

With the merits of easy synthesis, strong modifiability, and high affinity, aptamers have been broadly applied for protein targeting in bioanalysis, diagnosis, and therapeutics. The selection of protein-targeted aptamers is currently largely dependent on solid-liquid separation by using different types of nano- or micro-beads. However, the use of beads inescapably introduces unwanted nonspecific binding and thus affects selection efficiency. In order to sidestep this obstacle, we herein report an integrated technique to facilitate the discovery and development of protein-targeting aptamers by incorporating formaldehyde cross-linking with phase separation (FCPS). The feasibility and universality of FCPS were confirmed by the successful selection of two aptamers that could target various antibodies. Unlike traditional approaches, the proposed technique avoids the use of beads and enables the rapid generation of aptamers after only one to three rounds of selection. The as-selected aptamers were further used to regulate and control antibody activity, showing potential applications in biomedicine.

A multiparametric fluorescent visualization approach for detecting drug resistance in living cancer cells.

Drug resistance is a worldwide health care crisis which impedes disease treatment and increases financial burden, especially for its multifactorial nature and high complexity. Herein, we developed a multiparametric approach to visualize and detect drug resistance in living cancer cells, through the combination of DNA-templated covalent protein labeling strategy and fluorescent resonance energy transfer technique. Gefitinib resistance in non-small cell lung cancer caused by mesenchymal-epidermal transition factor (Met) overexpression and hyperactivation was investigated as a proof-of-concept. Unlike the traditional single-factor investigation, the proposed approach evaluated the contribution of three important parameters towards the resistance, including the changes of Met expression level, the homodimerization of Met with itself and the heterodimerization of Met with epidermal growth factor receptor (EGFR). A multiple regression model based on these three parameters was tentatively established for evaluation of the resistance level of laboratory-developed resistant cells and evaluation of the resistance level of patient-derived cells. Such an approach facilitates a quick identification of a drug resistance, to evaluate not only the resistance level but also the resistance mechanism.

Bioorthogonal Probes for Analyzing Non-Enzymatic Post-Translational Modifications.

Non-enzymatic post-translational modifications (nPTMs) have been proposed as indicators of cellular stresses and diseases. Unfortunately, direct assessment of nPTMs in native environment is extremely challenging due to the heterogeneity of adducts and the lack of tagging tools. Given these challenges, bioorthogonal probes (BPs) have been developed for the analysis of nPTMs. The rationality is that BPs could selectively install azides or alkynes into nPTMs as a biorthogonal handle for the following enrichment or tracking. Herein, we review the state-of-art of BPs used for nPTMs studies, clarify their working principles, and highlight how they advance our understanding of the biological functions of nPTMs.

The gut microbiota of gibbons across host genus and captive site in China.

Gut microbiota influences nutrient metabolism and immunity of animal hosts. Better understanding of the composition and diversity of gut microbiota contributes to conservation and management of threatened animals both in situ and ex situ. In this study, we applied 16S rRNA gene amplicon sequencing to evaluate the composition and diversity of the fecal bacterial community of four gibbon genera (Family Hylobatidae) at four Chinese zoos. The results showed that the dominant bacterial phyla were Bacteroidetes, Firmicutes, and Proteobacteria and dominant families were Prevotellaceae (Bacteroidetes), Spirochaetaceae (Spirochaetes) and Ruminococcaceae (Firmicutes) in the gut of all gibbons. Both captive site and host genus had significant effects on the relative abundance of dominant bacteria and structure of gut bacterial community. We found that captive site and host genus did not solely impact gut bacterial diversity, but the interaction between them did. This study provides basic knowledge for gut microbiota of all four gibbon genera and contributes to management and conservation of captive gibbons.

Rapid Noninvasive Skin Monitoring by Surface Mass Recording and Data Learning.

Skin problems are often overlooked due to a lack of robust and patient-friendly monitoring tools. Herein, we report a rapid, noninvasive, and high-throughput analytical chemical methodology, aiming at real-time monitoring of skin conditions and early detection of skin disorders. Within this methodology, adhesive sampling and laser desorption ionization mass spectrometry are coordinated to record skin surface molecular mass in minutes. Automated result interpretation is achieved by data learning, using similarity scoring and machine learning algorithms. Feasibility of the methodology has been demonstrated after testing a total of 117 healthy, benign-disordered, or malignant-disordered skins. Remarkably, skin malignancy, using melanoma as a proof of concept, was detected with 100% accuracy already at early stages when the lesions were submillimeter-sized, far beyond the detection limit of most existing noninvasive diagnosis tools. Moreover, the malignancy development over time has also been monitored successfully, showing the potential to predict skin disorder progression. Capable of detecting skin alterations at the molecular level in a nonsurgical and time-saving manner, this analytical chemistry platform is promising to build personalized skin care.

Tape-Stripping Electrochemical Detection of Melanoma.

A noninvasive electrochemical melanoma detection approach based on using adhesive tapes for collecting and fixing cells from a suspicious skin area and transferring the cells into a scanning electrochemical microscope (SECM) is presented. The adhesive layer collects the cells reproducibly and keeps them well adhered on the tape during experiments in an electrolyte solution. A melanoma biomarker, here the intracellular enzyme tyrosinase (TYR), was imaged on the tape-collected cells without further cell lysing using antibodies that were labeled with horseradish peroxidase (HRP). The HRP labels catalyzed the oxidation of a dissolved redox-active species, which was detected at a soft microelectrode, gently brushed in contact mode over the tape. The melanoma biomarker was first detected on tape-stripped samples with murine melanoma cells of different concentrations. Thereafter, increasing levels of TYR were recorded in cells that were collected from the skin of melanoma mouse models representing three different stages of tumor growth. Additionally, SECM results of tape-stripped different human melanoma cell lines were confirmed by previous studies based on traditionally fixed and permeabilized cells.

Immuno-affinity Amperometric Detection of Bacterial Infections.

A combination of an immuno-affinity enrichment strategy and sensitive amperometric read-out was implemented in a point-of-care platform intended for bacterial infection analysis. Bacterial cells, selectively captured and enriched from complex matrices through immuno-affinity, were detected by amperometric monitoring of the redox state of metabolic activity indicators, providing species identification and viable-cell quantification. The method was successfully employed for the diagnosis of bacterial infections including antimicrobial susceptibility testing with only several hours of total working time.

Detection of antimicrobial resistance-associated proteins by titanium dioxide-facilitated intact bacteria mass spectrometry.

Titanium dioxide-modified target plates were developed to enhance intact bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The plates were designed to photocatalytically destroy the bacterial envelope structure and improve the ionization efficiency of intracellular components, thereby promoting the measurable mass range and the achievable detection sensitivity. Accordingly, a method for rapid detection of antimicrobial resistance-associated proteins, conferring bacterial resistance against antimicrobial drugs, was established by mass spectrometric fingerprinting of intact bacteria without the need for any sample pre-treatment. With this method, the variations in resistance proteins' expression levels within bacteria were quickly measured from the relative peak intensities. This approach of resistance protein detection directly from intact bacteria by mass spectrometry is useful for fast discrimination of antimicrobial-resistant bacteria from their non-resistant counterparts whilst performing species identification. Also, it could be used as a rapid and convenient way for initial determination of the underlying resistance mechanisms.

Sensitive and fast identification of bacteria in blood samples by immunoaffinity mass spectrometry for quick BSI diagnosis.

Bloodstream infections rank among the most serious causes of morbidity and mortality in hospitalized patients, partly due to the long period (up to one week) required for clinical diagnosis. In this work, we have developed a sensitive method to quickly and accurately identify bacteria in human blood samples by combining optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and efficient immunoaffinity enrichment/separation. A library of bacteria reference mass spectra at different cell numbers was firstly built. Due to a reduced sample spot size, the reference spectra could be obtained from as few as 10 to 102 intact bacterial cells. Bacteria in human blood samples were then extracted using antibodies-modified magnetic beads for MS fingerprinting. By comparing the sample spectra with the reference spectra based on a cosine correlation, bacteria with concentrations as low as 500 cells per mL in blood serum and 8000 cells per mL in whole blood were identified. The proposed method was further applied to positive clinical blood cultures (BCs) provided by a local hospital, where Escherichia coli and Staphylococcus aureus were identified. Because of the method's high sensitivity, the BC time required for diagnosis can be greatly reduced. As a proof of concept, whole blood spiked with a low initial concentration (102 or 103 cells per mL) of bacteria was cultured in commercial BC bottles and analysed by the developed method after different BC times. Bacteria were successfully identified after 4 hours of BC. Therefore, an entire diagnostic process could be accurately accomplished within half a day using the newly developed method, which could facilitate the timely determination of appropriate anti-bacterial therapy and decrease the risk of mortality from bloodstream infections.

Novel dinorcassane- and cassane-type diterpenes from the seeds of Caesalpinia minax.

Two novel diterpenes, norcaesalpinin I (1) featuring an unusual ring C-contracted dinorcassane and caesalpinin U (2) possessing a highly oxygenated furanocassane skeleton were isolated from the seeds of Caesalpinia minax. Their structures were determined by different spectroscopic methods. A plausible biosynthetic pathway of 1 was proposed. The cytotoxic activity of compounds 1 and 2 against HepG2 and HeLa human tumor cell lines was evaluated.

Microfluidic chip integrated with flexible PDMS-based electrochemical cytosensor for dynamic analysis of drug-induced apoptosis on HeLa cells.

A novel microfluidic platform integrated with a flexible PDMS-based electrochemical cytosensor was developed for real-time monitoring of the proliferation and apoptosis of HeLa cells. The PDMS-gold film, which had a conductive smooth surface and was semi-transparent, facilitated electrochemical measurements and optical microscope observations. We observed distinct increases and decreases in peak current intensity, corresponding to cell proliferation in culture medium and apoptosis in the presence of an anticancer drug, respectively. This electrochemical analysis method permitted real-time, label-free monitoring of cell behavior, and the electrochemical results were confirmed with optical microscopy. The flexible microfluidic electrochemical platform presented here is suitable for on-site monitoring of cell behavior in microenvironments.

Fluorescent immunosensor based on CuS nanoparticles for sensitive detection of cancer biomarker.

A novel fluorescent immunosensor was developed based on the use of CuS nanoparticles (CuS NPs) as labels for the highly sensitive detection of human prostate cancer biomarker prostate specific antigen (PSA). In the presence of CuS NPs, the non-fluorescent substrate o-phenylenediamine could be oxidized into the stable fluorescent product 2,3-diamiophenazine at physiological pH. Throughout the reaction, no other oxidizing agents (e.g. hydrogen peroxide) were needed. The relatively mild oxidation conditions made the immunoassay robust, reliable and facile. The proposed immunoassay exhibited high sensitivity and specificity for the detection of PSA. A linear relationship between the fluorescent signals and the concentration of PSA was obtained in the range of 0.5 pg mL(-1) to 50 ng mL(-1), with a detection limit of 0.1 pg mL(-1) (S/N = 3). The proposed fluorescent immunoassay can be used as a promising platform for the detection of a variety of other biomarkers.

Ultrasensitive multianalyte electrochemical immunoassay based on metal ion functionalized titanium phosphate nanospheres.

A novel multianalyte electrochemical immunoassay was developed for ultrasensitive detection of human cardiopathy biomarkers cardiac troponin I (cTnI) and human heart-type fatty-acid-binding protein (FABP) using metal ion functionalized titanium phosphate nanospheres (TiP-metal ion) as labels. The metal ions could be detected directly through square wave voltammetry (SWV) without metal preconcentration, and the distinct voltammetric peaks had a close relationship with each sandwich-type immunoreaction. The position and size of the peaks reflected the identity and level of the corresponding antigen. The large amount of metal ions loading on the TiP nanospheres greatly amplified the detection signals, and the good biocompatibility of graphene nanoribbons (GONRs) retained good stability for the sandwich-type immunoassay. The proposed immunoassay exhibited high sensitivity and selectivity for the detection of cTnI and FABP. The linear relationships between electrochemical signals and the concentrations of cTnI and FABP were obtained in the range of 0.05 pg/mL-50 ng/mL and 0.05 pg/mL-50 ng/mL, respectively. The detection limits of cTnI and HIgG were 1 and 3 fg/mL (S/N = 3), respectively. Moreover, the immunoassay accurately detected the concentrations of cTnI and FABP in human serum samples, which were demonstrated to have excellent correlations with the standard enzyme linked immunosorbent assay (ELISA) method. The results suggested that the electrochemical immunoassay would be promising in the point-of-care diagnostics application of clinical screening of acute myocardial infarction (AMI) biomarkers.

Microfluidic platform for the evaluation of multi-glycan expressions on living cells using electrochemical impedance spectroscopy and optical microscope.

A microfluidic platform to evaluate the expression of multi-glycans on a cell surface was developed using electrochemical impedance spectroscopy (EIS) and optical microscope technique. In the microfluidic channel, four indium tin oxide (ITO) electrodes were modified with three lectins and one passivation agent, respectively, to selectively recognize the corresponding carbohydrate epitopes on the cell surface. The binding of the cells on the electrode array was monitored by the electrochemical impedance to evaluate the expression of cell surface glycans. The excellent optical transparency of ITO electrode permitted the microscopic observation of the cell binding simultaneously to substantiate the impedance measurement. Compared with the individual technology, the double-check mode increased the sensitivity and accuracy of the assay. The experimental results using these two techniques indicated that the cell binding ability decreased in the order WGA > Con A > PNA, which was consistent with the expression difference of carbohydrate epitopes on K562 cell surface. The proposed strategy was further used for facile evaluating the variations of glycan expression on living cells in response to drugs. The consumption of cell sample for each sensing interface in the whole experiments is merely 5 × 10(3) cells. This platform offers great promise for cancer-associated glycol-biomarkers screening and further helps cancer diagnosis and treatment.

Synthesis of Cd(2+)-functionalized titanium phosphate nanoparticles and application as labels for electrochemical immunoassays.

Porous titanium phosphate (TiP) nanoparticles with high water solubility have been synthesized by using the surfactant sodium dodecyl sulfate (SDS) as the structure-directing agent. The TiP nanoparticles can be functionalized with Cd(2+) and can be further used as labels for electrochemical detection of proteins due to their excellent ion-exchange property.