Highly-specific aptamer targeting SARS-CoV-2 S1 protein screened on an automatic integrated microfluidic system for COVID-19 diagnosis.

Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.

An integrated microfluidic platform for detection of ovarian clear cell carcinoma mRNA biomarker FXYD2.

In this work we developed an integrated microfluidic system for automatically detecting the ovarian clear cell carcinoma (OCCC) biomarker FXYD2. Dealing with ascites from ovarian cancer patients, capture of cancer cells, isolation of messenger RNA, and quantitative reverse-transcription polymerase chain reaction were integrated into a single microfluidic chip and carried out on-chip automatically. OCCC is a subtype of ovarian cancer with a high mortality risk, and a high FXYD2 gene expression level was shown to be closely associated with OCCC. The lowest limit of quantification using a benchtop protocol of this system could be as low as 100 copies per sample. By normalizing the expression to a housekeeping gene, GAPDH, a simple cycle threshold ratio index could distinguish high FXYD2 expression cells from the low-expression ones. This developed platform may therefore facilitate future OCCC diagnosis and/or prognosis.

Dual aptamer assay for detection of Acinetobacter baumannii on an electromagnetically-driven microfluidic platform.

Rapid detection of Acinetobacter baumannii (AB) is critical for limiting healthcare-associated infections and providing the best treatment for infected individuals. Herein an integrated microfluidic device for AB diagnosis utilizing a new dual aptamer assay was developed for point-of-care (POC) applications; magnetic beads coated with AB-specific aptamers were used to capture bacteria, and quantum dots (QD) bound to a second aptamer were utilized to quantify the amount of bacteria with a light-emitting diode (LED)-induced fluorescence module integrated into the device. Within a rapid detection of 30 min, a limit of detection of only 100 colony-forming units (CFU)/reaction was obtained, and all necessary microfluidic devices were actuated by a combination of permanent magnets and electromagnets. The pumping rate of the micropump was 270 µL/min at only 10 V, which is amenable for POC applications with lower power consumption, and only 10 µL of sample and reagents were required. Given these attributes, an automatic POC device was demonstrated which could perform a dual aptamer assay to diagnose AB by using electromagnetically-driven microfluidic system. This system provides a rapid, sensitive, low power and reagents consumption and fully automated for AB detection by using a dual aptamer assay. It will allow rapid clinical diagnosis of AB in the near future.

Optimization of an enzyme linked DNA aptamer assay for cardiac troponin I detection: synchronous multiple sample analysis on an integrated microfluidic platform.

In this study, an enzyme linked DNA aptamer based assay was optimized for human cardiac troponin I (cTnI) detection which is a prominent biomarker for acute myocardial infarction (AMI), on an integrated microfluidic platform. This platform allowed for the multiplex detection of six samples (5 µL per sample), and only 30 min were required for detection. First, cTnI-specific aptamers were surface-coated on magnetic beads. Bead-captured proteins were allowed to bind to a primary cTnI antibody and then to a secondary antibody labelled with horseradish peroxidase. Finally, chemiluminescence intensities were detected for quantification of cTnI. Purified proteins, serum from AMI patients and unknown serum samples were used to test the efficacy of the on-chip system. The limit of detection was measured to be only 12 ng L-1, and off-target effects from other proteins were minimal. This sensitive, cTnI-specific aptamer-based assay could consequently be used for reliable diagnosis of AMI.

Simultaneous detection of multiple NT-proBNP clinical samples utilizing an aptamer-based sandwich assay on an integrated microfluidic system.

Although cardiovascular diseases such as heart failure (HF) affect 30 million people globally, the early detection of HF has, until recently, been difficult and prone to misdiagnoses. Monitoring the circulatory levels of a relatively new biomarker, the N-terminal prohormone of a B-type natriuretic peptide, could be used for early risk evaluation of HF. Therefore, we developed a pneumatically-driven, automatic integrated microfluidic platform equipped with micromixers, micropumps, and microvalves for the simultaneous detection of NT-proBNP in up to six clinical samples within 25 min by using a novel aptamer-based sandwich assay, and the limit of detection was only 1.53 pg mL-1; given that the chip is 64% more compact than those developed in our prior works and requires only 5 µL of sample input, it may serve as a promising tool for early diagnosis of HF.

An integrated microfluidic system with field-effect-transistor sensor arrays for detecting multiple cardiovascular biomarkers from clinical samples.

Certain blood-borne biomarkers offer a potent methodology for understanding the risk of cardiovascular diseases (CVDs) with clinicians generally advocating the use of multiple biomarkers for proper risk assessment of CVDs. Herein four such CVDs biomarkers- C-reactive protein (CRP), N-terminal pro b-type natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI), and fibrinogen- were rapidly (5 min) analyzed from clinical samples (~ 4 µL) on an integrated microfluidic platform equipped with 1) immobilized highly specific aptamer probes and 2) field-effect transistor (FET)-based sensor arrays. The calibration curve from the FET sensor arrays showed good agreement in the physiological concentration ranges for CRP (0.1-50 mg/L), NT-proBNP (50-10,000 pg/mL), cTnI (1-10,000 pg/mL), and fibrinogen (0.1-5 mg/mL). The developed prototype of this fully automated portable device requires minimal reagent and sample inputs and consequently shows great promise for next-generation point-of-care devices assaying multiple CVDs biomarkers in clinical samples.

An integrated microfluidic platform to perform uninterrupted SELEX cycles to screen affinity reagents specific to cardiovascular biomarkers.

As cardiovascular diseases (CVD) are responsible for millions of deaths annually, there is a need for rapid and sensitive diagnosis of CVD at earlier stages. Aptamers generated by systematic evolution of ligands by exponential enrichment (SELEX) processes have been shown to be superior to conventional antibody-based cardiac biomarker detection. However, SELEX is a complicated, lengthy procedure requiring multiple rounds of extraction/amplification and well-trained personnel. To circumvent such issue, we designed an automated, miniaturized SELEX platform for the screening of aptamers towards three protein biomarkers associated with CVDs: N-terminal pro-peptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen. The developed microfluidic platform was equipped with microfluidic devices capable of sample transport and mixing along with an on-chip nucleic acid amplification module such that the entire screening process (5 rounds of selection in 8 h.) could be performed consecutively on a single chip while consuming only 35 µL of reagents in each cycle. This system may therefore serve as a promising, sensitive, cost-effective platform for the selection of aptamers specific for CVD biomarkers.

A microarray-based approach to evaluate the functional significance of protein-binding motifs.

Intracellular proteins comprise numerous peptide motifs that interact with protein-binding domains. However, using sequence information alone, the identification of functionally relevant interaction motifs remains a challenge. Here, we present a microarray-based approach for the evaluation of peptides as protein-binding motifs. To this end, peptides corresponding to protein interaction motifs were spotted as a microarray. First, peptides were titrated with a pan-specific binder and the apparent K(d) value of this binder for each peptide was determined. For phosphotyrosine-containing peptides, an anti-phosphotyrosine antibody was employed. Then, in the presence of the pan-specific binder, arrays were competitively titrated with cell lysate and competition constants were determined. Using the Cheng-Prusoff equation, binding constants for the pan-specific binder and inhibition constants for the lysates were converted into affinity constants for the lysate. We experimentally validate this method using a phosphotyrosine-binding SH2 domain as a further reference. Furthermore, strong binders correlated with binding motifs engaging in numerous interactions as predicted by Scansite. This method provides a highly parallel and robust approach to identify peptides corresponding to interaction motifs with strong binding capacity for proteins in the cell lysate.

Peptide microarrays to probe for competition for binding sites in a protein interaction network.

Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins will greatly overestimate connectivity within the network. Here, we addressed the impact of intracellular complexity on signalling networks using microarrays that carried a collection of peptides binding to the GRB2 SH2 and SH3 domains. Binding patterns and affinities for the recombinant adaptor protein GRB2 were compared with the ones for the protein in cell lysates. Peptide microarrays were titrated with the histidine-tagged recombinant protein, cell lysates or mixtures of both. Indeed, for recombinant GRB2, binding was detected for more peptides than for GRB2 in cell lysates. Moreover, binding was also observed for poor binders. It was impossible to define affinity thresholds for the binding of the recombinant protein to enable a discrimination of physiologically relevant interactions. Titrations of recombinant protein with lysate confirmed competition as the basis for fewer interactions. Importantly, the methods presented here enable the description of physiologically relevant binding patterns for proteins of interest and the identification of those peptide motifs, which are most strongly affected by competition. BIOLOGICAL SIGNIFICANCE: The biological significance of protein-protein interactions can only be addressed in a physiologically meaningful way in the presence of the endogenous proteome which may contain proteins that compete for binding sites. Using peptide microarrays, we here demonstrate for the adaptor protein GRB2 that this competition strongly reduces the number of interactions with other signalling proteins.

Analyzing the homeostasis of signaling proteins by a combination of Western blot and fluorescence correlation spectroscopy.

The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates. The concentration of the fusion protein is determined by fluorescence correlation spectroscopy, and this concentration is used to calibrate the intensity of bands on a Western blot. We applied this method to address cellular protein homeostasis by determining the concentrations of the plasma membrane-located transmembrane scaffolding protein LAT and soluble signaling proteins in naïve T cells and transformed T-cell lymphoma (Jurkat) cells (with the latter having nine times the volume of the former). Strikingly, the protein numbers of soluble proteins scaled with the cell volume, whereas that of the transmembrane protein LAT scaled with the membrane surface. This leads to significantly different stoichiometries of signaling proteins in transformed and naïve cells in concentration ranges that may translate directly into differences in complex formation.

Measurements of the intracellular stability of CPPs.

Nowadays, the analysis of the uptake and intracellular distribution of cell-penetrating peptides mostly relies on fluorescence microscopy, using fluorescently labeled CPP analogs. However, fluorescence microscopy does not reveal to which degree fluorescence reflects the intact peptide or only breakdown products. Here, we introduce fluorescence correlation spectroscopy (FCS) as a powerful method to address peptide stability in cells and cell lysates. Measurements in lysates of cells incubated with peptide yield information on degradation of the total cellular peptide content. In combination with protease inhibitors, such measurements enable conclusions on trafficking pathways. Intracellular FCS measurements provide direct information on peptide degradation and association with cellular structures in intact cells.

Simultaneous detection of intracellular target and off-target binding of small molecule cancer drugs at nanomolar concentrations.

BACKGROUND AND PURPOSE: In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem. EXPERIMENTAL APPROACH: The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1. KEY RESULTS: Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target. CONCLUSIONS AND IMPLICATIONS: The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.

Large-scale sequencing analysis of the full-length cDNA library of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the human cancers clearly linked to viral infections. Although the major risk factors for HCC development have been elucidated, the hepatocellular carcinogenesis pathway resulting in malignant transformation of liver cells remains to be clarified. Recently, some results of microarray and comparative genomic hybridization analysis have been provided as comprehensive studies of genomic instability in HCC, including mutation, deletion and DNA copy losses. In this work, the full-length cDNA library has been constructed and sequenced, and the sequencing results have been further clustered and analyzed. The results show that 1,342 genes have been found, and about 300 of these genes may be important in e.g. cell proliferation, DNA repair and apoptosis. After further analysis of DNA sequences, the deletion genotypes of at least 24 genes have been found. However, the functional changes of these deletion mutants and their significance in hepatocellular carcinogenesis remain to be clarified. This research may be one of the best to obtain the candidate genes for hepatocellular carcinogenesis.