Self-Assembled Pyrene Stacks and Peptide Monolayers Tune the Electronic Properties of Functionalized Electrolyte-Gated Graphene Field-Effect Transistors.

Aromatic molecules such as pyrenes are a unique class of building units for graphene functionalization, forming highly ordered π-π stacks while peptides provide more complex, biocompatible linkers. Understanding the adsorption and stacking behavior of these molecules and their influence on material properties is an essential step in enabling highly repeatable 2D material-based applications, such as biosensors, gas sensors, and solar cells. In this work, we characterize pyrene and peptide self-assembly on graphene substrates using fluorescence microscopy, atomic force microscopy and electrolyte-gated field-effect measurements supported by quantum mechanical calculations. We find distinct binding and assembly modes for pyrenes versus peptides with corresponding distinct electronic signatures in their characteristic charge neutrality point and field-effect slope responses. Our data demonstrates that pyrene- and peptide-based self-assembly platforms can be highly beneficial for precisely customizing graphene electronic properties for desired device technologies such as transport-based biosensing graphene field-effect transistors.

Clinical performance evaluation of a SARS-CoV-2 Rapid Antibody Test for determining past exposure to SARS-CoV-2.

OBJECTIVES: Due to the number of asymptomatic infections and limited access to high-performance antibody tests, the true prevalence and seropositivity of SARS-CoV-2 infection remains unknown. To fill this gap, the clinical performance of a point-of-care SARS-CoV-2 Rapid Antibody Assay, a chromatographic immunoassay for detecting IgM/IgG antibodies, in near patient settings was assessed. METHODS: Forty-two anti-SARS-Cov-2 positive (CoV+) and 92 anti-SARS-Cov-2 negative (CoV-) leftover samples from before December 2019 were assessed; the Elecsys® Anti-SARS-CoV-2 was used as the reference assay. Analytical specificity was tested using leftover samples collected before December 2019 from patients with common cold symptoms. RESULTS: The SARS-CoV-2 Rapid Antibody Test was 100.0% (95% CI 91.59-100.0) sensitive and 96.74% (95% CI 90.77-99.32) specific, with 0.00% assay failure rate. No cross-reactivity was observed against the common cold panel. Method comparison was additionally conducted by two external laboratories, using 100 CoV+ and 275 CoV- samples, also comparing whole blood versus plasma matrix. The comparison demonstrated 96.00% positive and 96.36% negative percent agreement for plasma with the Elecsys Anti-SARS-CoV-2 and 99.20% percent overall agreement between whole blood and EDTA plasma. CONCLUSION: The SARS-CoV-2 Rapid Antibody Test demonstrated similar performance to the manufacturer's data and a centralised automated immunoassay, with no cross-reactivity with common cold panels.

A fully integrated microfluidic platform for highly sensitive analysis of immunochemical parameters.

We present a novel fully integrated centrifugal microfluidic platform for highly sensitive immunoassays in point-of-care settings. The platform consists of a disposable cartridge containing structures for assay processing, a porous membrane and all dried reagents required for the analysis. Additionally, a blister containing a washing buffer is connected to a new aliquoting structure enabling the serial aliquoting of washing buffer for repetitive bound-free separation steps. The proof-of-concept for two immunoassays is shown in the cartridge with each requiring only 30 µL of whole blood or plasma as the sample material. The detection of the cardiac marker Troponin T with a functional sensitivity of 7.55 ng L-1 (cv = 10%) within 11 minutes is shown based on samples from ten donors which were measured with six breadboard instruments to prove the platform capability for highly sensitive measurements at diagnostic relevant concentrations. Furthermore an assay for the cardiac marker NT-proBNP (five donors, six instruments) with a time-to-result of 12 minutes demonstrates that high-titer analytes (43 to 16.566 ng L-1) can be measured as well. A method comparison of our platform with a state-of-the-art laboratory analyzer proves an excellent correlation of the measured analyte concentrations. All results are obtained from injection moulded cartridges and all components of the platform are compatible for mass production.

A new immunochemistry platform for a guideline-compliant cardiac troponin T assay at the point of care: proof of principle.

BACKGROUND: A multitude of troponin assays for the point-of-care (POC) have been developed showing a lack of analytical sensitivity and precision. We present a new platform solution for the high-sensitivity detection of cardiac troponin T (cTnT) in a 30 µL whole blood sample with a turnaround time of 11 min. METHODS: The immunoassay was completely run in a ready-to-use plastic disposable, a centrifugal microfluidic disc with fully integrated reagents. After the sample application, the assay was automatically processed by separating the cellular blood components via centrifugation, followed by incubation of a defined volume from the generated plasma with the immunoreagents. The fluorescence in the signal zone of a membrane was measured after its washing for the cTnT quantitation. RESULTS: A calibration curve, measured in whole blood samples spiked with native human cTnT, was generated covering a range up to a concentration of approximately 8300 ng/L. The lower detection limit was determined to be 3.0 ng/L. At a concentration of 14 ng/L, the 99th percentile value from the high-sensitivity cardiac troponin T (hs-cTnT) assay in the Elecsys® system, the imprecision (CV) was 3.8%. A CV profile indicated that the functional sensitivity for a CV <10% was 6.8 ng/L. The assay did not show any significant cross-reaction with human skeletal troponin T. We observed an excellent correlation with the hs-TnT Elecsys® assay for 49 clinical plasma samples (r=0.9744). CONCLUSIONS: The described technology shows that an analytical performance for a highly sensitive determination of cTnT can be achieved in a POC setting.

Development of a 1-microl scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy.

This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.