Protein-templated copper nanoclusters for fluorimetric determination of human serum albumin.

Copper nanoclusters (CuNCs) are attractive for their unique optical properties, providing sensitive fluorescent detection of several kinds of targets even in complex matrices. Their ability in growing on suitable protein and nucleic acid templates make CuNCs efficient optical reporters to be exploited in bioanalysis. In this work, we report the specific and sensitive determination of human serum albumin (HSA) in human serum (HS) and urine via CuNCs fluorescence. HSA is the most abundant protein in plasma, and plays a key role in the early diagnosis of serious pathological conditions such as albuminuria and albuminemia. Recently, HSA has become clinically central also as a biomarker to assess severity, progression, and prognosis of various cancers. We report the controlled and reproducible growth of CuNCs directly on the target analyte, HSA, which results in a fine dose-dependent fluorescent emission at 405 nm. The protocol is optimized in water, and then applied to serum and urine specimens, without matrix pretreatment. The method linearly responds within the whole concentration of clinical interest, with a sensitivity of 1.8 ± 0.1 × 10-3 g L-1 and 0.62 ± 0.03 × 10-3 g L-1 in serum and urine, respectively, and excellent reproducibility (CVav% ca. 3% for both). The assay is designed to have a single protocol working for both matrices, with recovery of 95% (HS) and 96% (urine). The stability of the fluorescence after CuNCs formation was tested over 3 days, displaying good results (yet higher in urine than in serum).

A self-correcting fluorescent assay of tyrosinase based on Fe-MIL-88B-NH2 nanozyme.

A self-correcting fluorescent assay of tyrosinase (TYR) was developed by utilization of Fe-MIL-88B-NH2 as a peroxidase-like nanozyme and a capture probe. Fe-MIL-88B-NH2 nanozyme was selected as an electron donor, and the oxidization product (dopamine-o-quinone) acts as an energy acceptor. First, TYR catalyzes the oxidation of tyramine hydrochloride to dopamine and then to dopamine-o-quinone. Second, Fe-MIL-88B-NH2 with intrinsic peroxidase-like activity decomposes H2O2 to produce ·OH radicals, which further accelerate the oxidation of dopamine to dopamine-o-quinone. Excessive H2O2 and ·OH radicals reduce the interferences from ascorbic acid at the same time providing a self-correcting ability. Dopamine-o-quinone reacts with -NH2 groups on the ligand of Fe-MIL-88B-NH2 through Michael reaction which results in fluorescence quenching. Under 365-nm excitation, the fluorescence emission intensity at 452 nm gradually decreased with increasing TYR concentration varying from 0 to 10 U mL-1. The linear range is from 1 to 5 U mL-1 and the detection limit is 0.05679 U mL-1. This self-correcting fluorescent assay of tyrosinase exhibits good sensitivity and selectivity which is also successfully applied for tyrosinase inhibitor detection. Schematic representation of fluorescent assay for tyrosinase determination based on Fe-MIL-88B-NH2 nanozyme. A self-correcting fluorescent assay for tyrosinase was developed based on the Fe-MIL-88B-NH2 nanozyme.

Expanding the Toolbox for Label-Free Enzyme Assays: A Dinuclear Platinum(II) Complex/DNA Ensemble with Switchable Near-IR Emission.

Switchable luminescent bioprobes whose emission can be turned on as a function of specific enzymatic activity are emerging as important tools in chemical biology. We report a promising platform for the development of label-free and continuous enzymatic assays in high-throughput mode based on the reversible solvent-induced self-assembly of a neutral dinuclear Pt(II) complex. To demonstrate the utility of this strategy, the switchable luminescence of a dinuclear Pt(II) complex was utilized in developing an experimentally simple, fast (10 min), low cost, and label-free turn-on luminescence assay for the endonuclease enzyme DNAse I. The complex displays a near-IR (NIR) aggregation-induced emission at 785 nm in aqueous solution that is completely quenched upon binding to G-quadruplex DNA from the human c-myc oncogene. Luminescence is restored upon DNA degradation elicited by exposure to DNAse I. Correlation between near-IR luminescence intensity and DNAse I concentration in human serum samples allows for fast and label-free detection of DNAse I down to 0.002 U/mL. The Pt(II) complex/DNA assembly is also effective for identification of DNAse I inhibitors, and assays can be performed in multiwell plates compatible with high-throughput screening. The combination of sensitivity, speed, convenience, and cost render this method superior to all other reported luminescence-based DNAse I assays. The versatile response of the Pt(II) complex to DNA structures promises broad potential applications in developing real-time and label-free assays for other nucleases as well as enzymes that regulate DNA topology.

Surface-Enhanced Raman Scattering Active Core-Shell Ag NPs@Carbon Dots with Enzyme-Mimicking Activities for Label-Free Measurement Cholesterol.

Serological-sensitive testing of cholesterol holds significant value in the fields of healthcare and clinical diagnosis. This study reports on the preparation of peroxidase-mimicking nanozymes through the wrapping of N, S-doped carbon dots (DCDs) on the surface of silver nanoparticles (Ag NPs@DCD). The shell-core structure of Ag NPs@DCD displays peroxidase-mimicking capability, with the potential to catalyze inactive Raman probe molecules into the Raman reporters. Furthermore, a "shell-isolated nanoparticles-enhanced Raman spectroscopy" structure exhibited an enhanced Raman signal of reporter molecules. Ag NPs@DCD were utilized to create a label-free SERS sensing system for high-performance detection of cholesterol in serum samples. These results demonstrate the potential of the novel nanozyme-based SERS approach for clinical diagnosis.

RNA demethylation-driven functional supramolecular structure for label-free detection of m6A modification eraser FTO in human breast tissues.

Fat mass and obesity-associated enzyme (FTO) can dynamically regulate N6-methyladenosine modification, and it is engaged in various cellular functions. Herein, we demonstrate the RNA demethylation-driven functional supramolecular structure for label-free detection of m6A modification eraser FTO in human breast tissues. The presence of FTO catalyzes the removal of methyl group in m6A, causing the cleavage of demethylated DNA by DpnII and the release of DNA primer. The resultant DNA primer hybridizes with circular template to initiate isothermal rolling circle amplification (RCA), producing abundant long ssDNA polymers with repeating sequences of G-quadruplex. Subsequently, N-methylmesoporphyrin IX (NMM) is selectively embedded into G-quadruplex DNAzyme to form a supramolecular NMM-G-quadruplex structure for the generation of an amplified fluorescence signal. Benefiting from high selectivity of DpnII toward demethylated DNA, high amplification efficiency of RCA, and high signal-to-noise ratio of G-quadruplex-NMM system, this assay can sensitively detect FTO with a limit of detection (LOD) of 3.10 × 10-16 M, screen RNA demethylase inhibitors, quantify FTO activity in cancer cells, and discriminate FTO activity between breast cancer patient tissues and healthy person tissues. Importantly, this assay can be homogeneously conducted in a label-free manner, with great potential in RNA demethylases-related pathogenesis research and clinical diagnostics.

An alternative label-free DNA sensor based on the alternating-current electroluminescent device for simultaneous detection of human immunodeficiency virus and hepatitis C co-infection.

Coinfection of HIV/HCV is a significant public health issue globally, as it increases the risk of liver cancer in co-infected individuals. The point-of-care testing (POCT) device for HIV/HCV DNA detection is promptly needed for diagnosis and monitoring of the disease progression. Here, the alternating-current electroluminescence (ACEL) technique is proposed as a sensitive POCT sensing platform for HIV/HCV cDNA detection. A conductance-based light emission modulated by the hybridization between a pyrrolidinyl PNA probe and the DNA target enabled the DNA detection in a label-free format. Enhanced electroluminescence was observed in the presence of the target DNA due to the increased proton conductivity. Under the optimal conditions, the linearity range from 1 nM to 1 µM was achieved for HIV and HCV cDNA with LODs of 1.86 pM (HIV cDNA) and 1.96 pM (HCV cDNA). The spiked HIV/HCV cDNA in healthy human serum was successfully detected, demonstrating the feasibility of the developed device for the detection of cDNA in real biological samples. Additionally, simultaneous HIV/HCV cDNA detection on a single ACEL device employing a 2x2-array detection zone design. The cross-reactivity with other viral DNA was shown to be minimal due to the high specificity of the PNA probes used. Finally, the negative and positive samples from the patient's serum were tested and the results were in 100% agreement with the commercial kit based-on real-time PCR method, thus illustrating the high sensitivity and specificity of the developed sensor.

Design and pharmacological profile of a novel covalent partial agonist for the adenosine A1 receptor.

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A1 receptor (hA1AR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hA1AR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hA1AR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study. In a radioligand binding assay, LUF7746's apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hA1AR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hA1AR demonstrated that amino acid Y2717.36 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746's irreversible activation of an A1 receptor-mediated cell morphological response. These results led us to conclude that LUF7746 is a novel covalent hA1AR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist.

Label-free assay of protein kinase A activity and inhibition in cancer cell using electrochemically-prepared AuNP/rGO nanohybrid electrode modified with C-Kemptide.

A simple, label-free and sensitive electrochemical assay is described for the detection of protein kinase A (PKA) activity and inhibition in cancer cell by measuring the change in electrochemical impedance upon phosphorylation. The assay utilized gold nanoparticle (AuNP) and reduced graphene oxide (rGO) nanohybrid which was synthesized and deposited on the electrode from GO and Au3+ precursors by one-pot electrochemical synthesis. As-prepared AuNP/rGO electrode was employed to immobilize C-Kemptide peptide substrates which was phosphorylated in the presence of PKA and ATP. The resulting assay allowed effective, selective and sensitive monitoring of PKA activity according to the impedimetric change in the range of 0.1-500 U/mL, and the detection limit (LOD) is 53 mU/mL. It could also be used for the screening of protein kinase inhibitors. Furthermore, the assay could be applied for the evaluation of PKA activity and inhibition in HeLa cell samples. Therefore, the proposed assay provides one promising tool for PKA activity detection and inhibitor screening with excellent performance.

One-step label-free chemiluminescent assay for determination of exonuclease III activity towards hairpin oligonucleotides.

Fast label-free chemiluminescent assay for determination of exonuclease III (ExoIII) activity measured towards hairpin oligonucleotide substrates was developed. The designed substrates consisted of EAD2 aptamer to hemin which was associated with DNA sequence complementary to 5'-terminus fragment of EAD2. In the presence of ExoIII the associated sequence of the hairpin stem was digested, producing EAD2 aptamer which reacted with hemin with the formation of peroxidase-mimicking DNAzyme (PMDNAzyme). The catalytic activity of the produced PMDNAzyme was measured towards luminol/H2O2. Under the optimized conditions the limit of detection and sensitivity of the one-step chemiluminescent assay of ExoIII were 7.3 nM and 1.7 × 108 M-1, respectively. The coefficient of variation (CV) was lower than 6%.

Rapid Differentiation of Host and Parasitic Exosome Vesicles Using Microfluidic Photonic Crystal Biosensor.

Parasite extracellular vesicles (EVs) are potential biomarkers that could be exploited for the diagnosis of infectious disease. This paper reports a rapid bioassay to discriminate parasite and host EVs. The EV detection assay utilizes a label-free photonic crystal (PC) biosensor to detect the EVs using a host-specific transmembrane protein (CD63), which is present on EV secreted by host cells (modeled by murine macrophage cell line J774A.1) but is not expressed on EV secreted by parasitic nematodes such as the gastrointestinal nematode Ascaris suum. The surface of PC is functionalized to recognize CD63, and is sensitive to the changes in refractive index caused by the immobilization of EVs. The biosensor demonstrates a detection limit of 2.18 × 109 EVs/mL and a capability to characterize the affinity constants of antibody-host EV bindings. The discrimination of murine host EVs from parasite EVs indicates the capability of the sensor to differentiate EVs from different origins. The label-free, rapid EV assay could be used to detection parasite infection and facilitate the exosome-based clinic diagnosis and exosome research.

Robust real-time cell analysis method for determining viral infectious titers during development of a viral vaccine production process.

The classical cell-culture methods, such as cell culture infectious dose 50% (CCID50) assays, are time-consuming, end-point assays currently used during the development of a viral vaccine production process to measure viral infectious titers. However, they are not suitable for handling the large number of tests required for high-throughput and large-scale screening analyses. Impedance-based bio-sensing techniques used in real-time cell analysis (RTCA) to assess cell layer biological status in vitro, provide real-time data. In this proof-of-concept study, we assessed the correlation between the results from CCID50 and RTCA assays and compared time and costs using monovalent and tetravalent chimeric yellow fever dengue (CYD) vaccine strains. For the RTCA assay, Vero cells were infected with the CYD sample and real-time impedance was recorded, using the dimensionless cell index (CI). The CI peaked just after infection and decreased as the viral cytopathic effect occurred in a dose-dependent manner. The time to the median CI (CITmed) was correlated with viral titers determined by CCID50 over a range of about 4-5log10 CCID50/ml. This in-house RTCA virus-titration assay was shown to be a robust method for determining real-time viral infectious titers, and could be an alternative to the classical CCID50 assay during the development of viral vaccine production process.

Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies.

Neuropeptide S (NPS) is the endogenous ligand of the neuropeptide S receptor (NPSR). NPS modulates several biological functions including anxiety, wakefulness, pain, and drug abuse. The aim of this study was the investigation of the pharmacological profile of NPSR using the dynamic mass redistribution (DMR) assay. DMR is a label-free assay that offers a holistic view of cellular responses after receptor activation. HEK293 cells stably transfected with the murine NPSR (HEK293mNPSR) have been used. To investigate the nature of the NPS-evoked DMR signaling, FR900359 (Gq inhibitor), pertussis toxin (Gi inhibitor), and rolipram (phosphodiesterase inhibitor) were used. To determine the pharmacology of NPSR, several selective ligands (agonists, partial agonists, antagonists) have been tested. NPS, through selective NPSR activation, evoked a robust DMR signal with potency in the nanomolar range. This signal was predominantly, but not completely, blocked by FR900359, suggesting the involvement of the Gq-dependent signaling cascade. NPSR ligands (agonists and antagonists) displayed potency values in DMR experiments similar, but not identical, to those reported in the literature. Furthermore, partial agonists produced a higher efficacy in DMR than in calcium experiments. DMR can be successfully used to study the pharmacology and signaling properties of novel NPSR ligands. This innovative approach will likely increase the translational value of in vitro pharmacological studies.

Piezoelectric biosensor for the determination of Tumor Necrosis Factor Alpha.

Tumor Necrosis Factor Alpha (TNFα) is an important marker of inflammatory processes in human body. In the current healthcare, determination of TNFα blood or plasma level is done by Enzyme Linked Immuno-Sorbent Assay (ELISA) as a primary choice method. Piezoelectric immunosensors are analytical platform recording affinity interactions on their surface. It is inferred that the immunosensors would be a functional alternative to the ELISA. In this study, antibody against TNFα was immobilized on Quartz Crystal Microbalance (QCM) sensor and the same was made on magnetic particles. Human TNFα was measured in a way of interaction with QCM surface and then the particles were applied. The assay exerted sufficient limit of detection equal to 1.62pg/ml and it also fully correlated with standard ELISA tests. No interference by interleukin 6 or albumin was observed. Long term stability of the immunosensors lasting for at least three months was found. The immunosensors appears to be readily for practical performance and it would be an alternative to the standard ELISA especially when diagnoses made in field, homecare conditions or conditions of small hospitals as an emergency test.

Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils.

INTRODUCTION: The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors (FPRs) in human neutrophils. METHODS: Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist potencies. RESULTS: The potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H. DISCUSSION: We conclude that the DMR assay can be used, and complements more traditional methods, to study the signalling and pharmacology of endogenous FPR receptors in human neutrophils.

Label-Free Biosensor Detection of Endocrine Disrupting Compounds Using Engineered Estrogen Receptors.

Endocrine Disrupting Compounds (EDCs) are chemical substances shown to interfere with endogenous hormones affecting the endocrine, immune and nervous systems of mammals. EDCs are the causative agents of diseases including reproductive disorders and cancers. This highlights the urgency to develop fast and sensitive methods to detect EDCs, which are detrimental even at very low concentrations. In this work, we propose a label-free surface plasmon resonance (SPR) biosensor method to detect specific EDCs (17 ß-estradiol (E2), ethinyl-estradiol, 4-nonylphenol, tamoxifen) through their binding to estrogen receptor alpha (ERα). We show that the use of rationally designed ERα (as bio-recognition element) in combination with conformation-sensitive peptides (as amplification agent, resulting in increased responses) enables the detection of low parts per billion (ppb) levels of E2. As a proof of concept, this bioassay was used to detect E2 in (spiked) real water samples from fish farms, rivers and the sea at low ppb levels after concentration by solid phase extraction. In addition, the present SPR assay that combines a conformation-sensitive peptide with an array of ERα mutants is very promising for the assessment of the risk of potential estrogenic activity for chemical substances.

Fragment-Based Screening for Enzyme Inhibitors Using Calorimetry.

Isothermal titration calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of binding reactions. In addition, it enables label-free measurement of enzymatic reactions. The advent of extremely sensitive microcalorimeters have made it increasingly valuable as a tool for hit validation and characterization, but its use in primary screening is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional ITC, particularly for screening libraries of 500-1000 compounds such as those encountered in fragment-based lead discovery. This chapter describes how nanocalorimetry and conventional microcalorimetry can be used to screen compound libraries for enzyme inhibitors.

Microfluidic immunoassay with plug-in liquid crystal for optical detection of antibody.

Recent advance in liquid crystal (LqC) based immunoassays enables label-free detection of antibody, but manual preparation of LqC cells and injection of LqC are required. In this work, we developed a new format of LqC-based immunoassay which is hosted in a microfluidic device. In this format, the orientations of LqC are strongly influenced by four channel walls surrounding the LqC. When the aspect ratio (depth/width) of the channel is smaller than 0.38, LqC orients homeotropically inside the microchannel and appears dark. After antigens bind to immobilized antibodies on the channel walls, a shift of the LqC appearance from dark to bright (due to the disruption of LqC orientation) can be visualized directly. To streamline the immunoassay process, a tubing cartridge loaded with a sample solution, washing buffers and a plug of LqC is connected to the microfluidic device. By using pressure-driven flow, the cartridge allows antigen/antibody binding, washing and optical detection to be accomplished in a sequential order. We demonstrate that this microfluidic immunoassay is able to detect anti-rabbit IgG with a naked-eye detection limit down to 1 µg mL(-1). This new format of immunoassay provides a simple and robust approach to perform LqC-based label-free immunodetection in microfluidic devices.

Identification and optimization of PDE10A inhibitors using fragment-based screening by nanocalorimetry and X-ray crystallography.

Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.

Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs).

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5-10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 µl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R(2)=0.990, which was comparable to the value of R(2)=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.