Clinical Bioanalysis Research Articles

Luminescent Terbium Probe for Time‐Resolved FRET and NSET Binding Assays with Quantum Dots and Gold Nanoparticles

Time‐gated or time‐resolved FRET (TR‐FRET) assays are important tools in biosensing, bioimaging, drug screening, and molecular diagnostics. Efficient TR‐FRET assays require stable lanthanide complexes with high absorption cross sections, high quantum yields, and long photoluminescence lifetimes. Owing to their challenging synthesis, such complexes are relatively rare and new components are of potential interest when developing TR‐FRET probes. Here, we evaluate the recently developed Tb complex CoraFluor‐1 concerning its analytical performance in terbium‐to‐quantum dot FRET and terbium‐to‐gold nanoparticle NSET assays using the prototypical biological recognition system of biotin and streptavidin. Biological binding was quantifiable at sub‐picomolar concentrations in small sample volumes, with broad applicability demonstrated across three commercial fluorescence plate readers used for time‐resolved, spectrally‐resolved, and clinical bioanalysis. Overall, CoraFluor‐1 provided excellent analytical performance as both FRET and NSET donor, validating its potential for developing new TR‐FRET probes for biosensing and bioimaging.

Synthesis of Stable Isotope, Tritiated, and Carbon-14 Labeled Balcinrenone.

As part of a medicinal chemistry program aimed at discovering a mineralocorticoid receptor modulator for treatment of kidney and cardiovascular indications, multiple labeled versions of the lead compound, balcinrenone (AZD9977), were prepared. Four stable isotope labeled versions of the compound were prepared for clinical bioanalysis and biological investigations. Three of these stable isotope labeled compounds were tritiated as well as the parent for biology applications and DMPK investigations. They were prepared using a standard iodination-tritiodehalogentation approach. Finally, AZD9977 was prepared in carbon-14 labeled form for preclinical and clinical applications.

Room temperature roll-to-roll additive manufacturing of polydimethylsiloxane-based centrifugal microfluidic device for on-site isolation of ribonucleic acid from whole blood

Polymer-based lab-on-a-disc (LoaD) devices for isolating ribonucleic acid (RNA) from whole blood samples have gained considerable attention for accurate biomedical analysis and point-of-care diagnostics. However, the mass production of these devices remains challenging in manufacturing cost and sustainability, primarily due to the utilization of a laser cutter or router computer numerical control (CNC) machine for engraving and cutting plastics in the conventional prototyping process. Herein, we reported the first energy-efficient room-temperature printing-imprinting integrated roll-to-roll manufacturing platform for mass production of a polydimethylsiloxane (PDMS)-based LoaD to on-site isolate ribonucleic acid (RNA) from undiluted blood samples. We significantly reduced energy consumption and eliminated thermal expansion variations between the mold, substrate, and resists by accelerating the PDMS curing time to less than 10 min at room temperature without using heat or ultraviolet radiation. The additive manufacturing technology was applied to fabricate a multi-depth flexible polymer mold that integrated macro (2 mm) and micro-sized (500 μm) features, which overcomes the economic and environmental challenges of conventional molding techniques. Our integrated R2R platform was enabled to print adhesion-promoting films at the first printing unit and continuously in-line imprint with a high replication accuracy (99%) for high-volume manufacturing of a new centrifugal microfluidic chip with an enhancement of mixing performance by integrating an efficient mixing chamber and serpentine micromixer. This research paved the way for scalable green manufacturing of large-volume polymer-based microfluidic devices, often required in real-world sample-driven analytical systems for clinical bioanalysis.

Effect of non-surgical periodontal treatment on cytokines/adipocytokines levels among periodontitis patients with or without obesity: a systematic review and meta-analysis

BackgroundThe objective of this systematic review and meta-analysis was to evaluate the effects of non-surgical periodontal therapy (NSPT) on inflammatory-related cytokines/adipocytokines in periodontitis patients with or without obesity.MethodsWe followed the preferred reporting items for systematic reviews and meta-analyses statement and registered the study (CRD42022375331) in the Prospective International Register of Systematic Reviews. We screened randomized-controlled trials and controlled clinical trials from six databases up to December 2022. Quality assessment was performed with RoB-2 and ROBINS-I tools for randomized trials and non-randomized trials, respectively. Meta-analysis was carried out using a random-effect model.ResultsWe included seventeen references in the systematic analysis, and sixteen in the meta-analysis. Baseline results of pro-inflammatory biomarkers, including serum interleukin (IL)-6, serum and gingival crevicular fluid (GCF), tumor necrosis factor (TNF)-a, serum C-reactive protein (CRP)/hs-CRP, and serum and GCF resistin, were higher in obesity subjects than in normal weight subjects. The effect of NSPT with respect to levels of cytokines/adipocytokines, including IL-6, TNF-a, CRP/hs-CRP, resistin, adiponectin, leptin and retinol binding protein 4 (RBP4), were then analyzed in the systematic and meta-analysis. After three months of NSPT, serum (MD = -0.54, CI = -0.62 – -0.46), and GCF (MD = -2.70, CI = -4.77 – -0.63) levels of IL-6, along with the serum RBP4 (MD = -0.39, CI = -0.68–0.10) decreased in periodontitis individuals with obesity. NSPT also improved GCF adiponectin levels after three months (MD = 2.37, CI = 0.29 – 4.45) in periodontitis individuals without obesity.ConclusionsObese status altered the baseline levels of cytokines/adipocytokines (serum IL-6, serum and GCF TNF-a, serum CRP/hs-CRP, and serum and GCF resistin). Then NSPT can shift the levels of specific pro-inflammatory mediators and anti-inflammatory mediators in biological fluids, both in obesity and non-obesity individuals. NSPT can reduce serum and GCF IL-6 levels together with serum RBP4 level in individuals with obesity after 3 months, besides, there is no sufficient evidence to prove that obese patients have a statistically significant decrease in the levels of other cytokines compared to patients with normal weight. NSPT can also increase GCF adiponectin level in normal weight individuals after 3 months. Our findings imply the potential ideal follow-up intervals and sensitive biomarkers for clinical bioanalysis in personalized decision-making of effect of NSPT due to patients’ BMI value.

A simple, sensitive, label-free electrochemical immunosensor based on the chitosan-coated silver/cerium oxide (CS@Ag/CeO2) nanocomposites for the detection of alpha-fetoprotein (AFP)

Metal oxide-based sensors have the benefit of inexpensive, quick response, and high sensitivity in detecting specific biological species. In this article, a simple electrochemical immunosensor was fabricated using antibody-chitosan coated silver/cerium oxide (Ab-CS@Ag/CeO2) nanocomposites on a gold electrode for sensitive alpha-fetoprotein (AFP) diagnosis in human serum samples. Successfully synthesis of AFP antibody-CS@Ag/CeO2 conjugates was confirmed through Fourier transform infrared spectra of the prototype. The amine coupling bond chemistry was then used to immobilize the resultant conjugate on a gold electrode surface. It was observed that the interaction of the synthesized Ab-CS@Ag/CeO2 nanocomposites with AFP prevented an electron transfer and reduced the voltammetric Fe(CN)6 3−/4− peak current, which was proportional to the amount of AFP. The linear ranges of AFP concentration were found from 10−12–10−6 g.ml−1. The limit of detection was calculated using the calibration curve and came out to be 0.57 pg.ml−1. The designed label-free immunosensor successfully detected AFP in human serum samples. As a result, the resulting immunosensor is a promising sensor plate form for AFP detection and could be used in clinical bioanalysis.

A sandwich-type electrochemical immunosensor for sensitive detection of HER2 based on dual signal amplification of La-MOF-PbO2 and PEI-MoS2NFs composites

In recent decades, the incidence of breast cancer has increased year by year, posing a serious threat to human health and quality of life, and about 30% of breast cancer patients have human epidermal growth factor receptor 2 (HER2) overexpression. Therefore, HER2 has become an important biomarker and indicator for the clinical evaluation of breast cancer in diagnosis, prognosis and recurrence. In this work, polyethyleneimine functionalized MoS2 nanoflowers (PEI-MoS2NFs) with good electrical conductivity and abundant active binding sites were designed and employed as a sensing platform for immobilizing the primary antibody of HER2 (Ab1). In addition, a La-MOF-PbO2 composite with a large specific surface area and good conductivity was used to load lots of electroactive toluidine blue (TB) and the secondary antibody of HER2 (Ab2) via gold nanoparticles (AuNPs) as the linker. Thus, the constructed sandwich-type electrochemical immunosensor was applied for sensitive detection of HER2, which showed a wide linear range from 100 fg mL−1 to 10 μg mL−1 with a lower limit of detection of 15.64 fg mL−1. Therefore, the resulting immunosensor in this study would have a potential application in clinical bioanalysis.

Volumetric Absorptive Microsampling in Therapeutic Drug Monitoring of Immunosuppressive Drugs-From Sampling and Analytical Issues to Clinical Application.

Miniaturisation and simplification are novel approaches in clinical bioanalysis, especially in therapeutic drug monitoring (TDM). These contemporary trends are related to the sampling, pre-treatment, and analysis of biological fluids. Currently, dried blood spot (DBS), one of the most popular microsampling techniques, is feasible and inexpensive. However, obtaining reliable results with sample homogeneity and volume variability is difficult. Volumetric Absorptive Microsampling (VAMS) has recently enabled the accurate and precise collection of a fixed blood volume. It reduced the hematocrit effect, improved volumetric accuracy, and generated results correlating with the dose and drug exposure from wet blood. This review focuses on VAMS-Mitra™ devices, which have become increasingly important since 2014, mainly for TDM and toxicology studies. First, the current literature has been reviewed based on immunosuppressants and their determination in samples obtained using Mitra™. Second, the critical points, weaknesses, and strengths have been characterized in contrast to classic venipuncture and other microsampling methods. Finally, we indicate the points of attention according to the perspective of Mitra™ as well as its usefulness in clinical practice. VAMS is currently state-of-the-art in microsampling and seems to be a good instrument for improving adherence to immunosuppressive therapy, especially in the pediatric population.

High efficiency electrochemiluminescence of 3D porous g-C3N4 with dissolved O2 as co-reactant and its sensing application for ultrasensitive detection of microRNA in tumor cells

In this work, the electrochemiluminescence (ECL) phenomenon of three-dimensional graphitic carbon nitride (3D g-C3N4) was reported. Firstly, the proposed 3D g-C3N4 possessed 3D porous interconnected open-framework which enabled faster charge transport and efficient penetration of co-reactants due to “pore confinement effect”. Then, we found that the dissolved O2 could serve as an excellent co-reactant for cathodic ECL of 3D g-C3N4. And the high specific surface area was beneficial to better adsorbing and gathering of dissolved O2 and reactive oxygen species (ROSs), which made them full contact on the surface or inside of 3D g-C3N4, giving a more sufficient ECL reaction and higher ECL signal. Based on the proposed 3D g-C3N4-O2 ECL system, a sensitive biosensor was constructed for microRNA-21 (miRNA-21) detection with assistance of 3D spherical tracks assisted 3D DNA walking machine, which exhibited superior performance for miRNA-21 with detection limit of 0.22 fM. The proposed 3D g-C3N4-O2 ECL system with high ECL efficiency and the effective target conversion and amplification strategies were beneficial to construct ultra-sensitive ECL sensing platform, which would be better applied to clinical bioanalysis.

Design and synthesis of a novel ZB716-d6 as a stable isotopically labeled internal standard

ZB716 is a synthetic, steroidal, orally active anti-estrogen agent that is under clinical development for the treatment of estrogen receptor (ER)-positive metastatic breast cancer. The stable isotope-labeled ZB716 was required for use as an internal standard in LC-MS/MS assays. Therefore, a novel deuterated ZB716 (ZB716-d6) as an isotopically labeled internal standard was designed and synthesized through a newly developed route, which prepared ZB716-d6 in eight steps from the commercially available deuterium-labeled starting material [2H6]pentafluoropentanol. This procedure is very practicable and gives the final compound in good yield (19% total yield) and high purity (D, >99%, chemical purity 98%). At present, ZB716-d6 has been successfully used as an internal standard in clinical bioanalysis.

Simultaneous Quantitation of Clevidipine and Its Active Metabolite H152/81 in Human Whole Blood by LC-MS/MS: Application to Bioequivalence Study.

Clevidipine is an ultrashort-acting dihydropyridine calcium antagonist, which can control blood pressure accurately. It is necessary to develop a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to quantitate clevidipine and its active metabolite H152/81 for clinical pharmacokinetic study and therapeutic drug monitoring. Liquid–liquid extraction was used for sample preparation, and clevidipine-d7 and H152/81-13C-d3 were chosen as the isotope internal standard. The chromatographic separation was performed on an ACE Excel 2 Phenyl column (50 × 2.1 mm). Mass quantification was carried out on the multiple reaction monitoring of the transitions of m/z 473.1→338.1, 480.1→338.1, 356.0→324.0, and 362.2→326.2 for clevidipine, clevidipine-d7, H152/81, and H152/81-13C-d3. The validated method gave an excellent linearity over a concentration range of 0.1–30 ng/ml for clevidipine and 2–600 ng/ml for H152/81. Other fully validated content such as accuracy, precision, extraction recovery, matrix effect, and stability were also investigated and showed satisfactory results. It was strongly recommended that whole blood is the first choice for clinical bioanalysis. Using whole blood for sample analysis can reduce the whole blood collection volume (1 ml vs. 4 ml) and shorten the time from sample collection to storage to 5 min, and there is no centrifugation process and precooling in the ice water bath, which can further reduce the instability caused by exposure. The method was successfully applied to a bioequivalence study of clevidipine butyrate-injectable emulsion.

Electrochemiluminescence Enhanced by the Synergetic Effect of Porphyrin and Multi‐walled Carbon Nanotubes for Uric Acid Detection

AbstractThe design and exploration of efficient, stable and environmentally compatible organic emitters for an electrochemiluminescence (ECL) sensor is a promising topic. Herein, a novel environmentally‐friendly luminophore, ZnBCBTP@MWCNTs, were fabricated via self‐assembly of porphyrin molecules (ZnBCBTP) onto multi‐walled carbon nanotubes (MWCNTs). The resulting luminophore ZnBCBTP@MWCNTs displayed not only the highly ECL property and but also the good accelerated electron mobility. Then, a label‐free ECL biosensor based ZnBCBTP@MWCNTs was constructed for the ultrasensitive detection of uric acid. Excitingly, this proposed ECL biosensor performed a good linear relationship in the range of 0–300 μM with a low detection limit of 1.4 μM, thus offering another reliable and feasible sensing platform for clinical bioanalysis with good selectivity, stability, and repeatability.

Entropy-driven amplified electrochemiluminescence biosensor for RdRp gene of SARS-CoV-2 detection with self-assembled DNA tetrahedron scaffolds

Dependable, specific and rapid diagnostic methods for severe acute respiratory syndrome β-coronavirus (SARS-CoV-2) detection are needed to promote public health interventions for coronavirus disease 2019 (COVID-19). Herein, we have established an entropy-driven amplified electrochemiluminescence (ECL) strategy to detect the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2 known as RdRp-COVID which as the target for SARS-CoV-2 plays an essential role in the diagnosis of COVID-19. For the construction of the sensors, DNA tetrahedron (DT) is modified on the surface of the electrode to furnish robust and programmable scaffolds materials, upon which target DNA-participated entropy-driven amplified reaction is efficiently conducted to link the Ru (bpy)32+ modified S3 to the linear ssDNA at the vertex of the tetrahedron and eventually present an “ECL on” state. The rigid tetrahedral structure of the DT probe enhances the ECL intensity and avoids the cross-reactivity between single-stranded DNA, thus increasing the sensitivity of the assays. The enzyme-free entropy-driven reaction prevents the use of expensive enzyme reagents and facilitates the realization of large-scale screening of SARS-CoV-2 patients. Our DT-based ECL sensor has demonstrated significant specificity and high sensitivity for SARS-CoV-2 with a limit of detection (LOD) down to 2.67 fM. Additionally, our operational method has achieved the detection of RdRp-COVID in human serum samples, which supplies a reliable and feasible sensing platform for the clinical bioanalysis.

РОЛЬ ВНЕДРЕНИЯ ЦИФРОВЫХ ПРОИЗВОДСТВЕННЫХ СИСТЕМ НА ФАРМАЦЕВТИЧЕСКОМ ПРЕДПРИЯТИИ

Due to the growing global crisis against the background of the COVID-19 pandemic, the issue of uninterrupted provision of medicines to the population has become critically important. In this regard, the introduction of information management systems at pharmaceutical enterprises is becoming particularly relevant. The pharmaceutical industry as a whole faces a number of unique challenges, including close regulatory oversight, sophisticated testing methods, and growing financial pressures as competition in the market increases. Industry leaders are digitizing core functions within their internal vertical operational processes, as well as with their value chain partners. One of the information management systems in the laboratory is the LIMS (Laboratory information system) information system. One of the challenges when choosing LIMS in pharmaceuticals is the variety of laboratories in the company - combinatorial chemistry, screening, preclinical and clinical bioanalysis, analytical chemistry, industrial R&D and production quality control, requiring unique needs and workflows. With the development of business, information technology and the regulatory framework, the role of LIMS in the pharmaceutical organization is also increasing. Proper implementation of this mechanism in the work of a pharmaceutical enterprise will help to preserve the quality of the product, reduce and optimize the work and satisfy the end user and government agencies.

Monoclonal Antibody Against Human IgG (Anti-Human IgG).

Monoclonal Antibodies in Immunodiagnosis and ImmunotherapyVol. 39, No. 5 Monoclonal AntibodiesMonoclonal Antibody Against Human IgG (Anti-Human IgG)Ruiyin ChuRuiyin ChuAddress correspondence to: Ruiyin Chu, Biomarker and Clinical Bioanalysis, Sanofi North America, Framingham, MA 01701, USA E-mail Address: ruiyin.chu@sanofi.comSearch for more papers by this authorPublished Online:21 Oct 2020https://doi.org/10.1089/mab.2020.0034AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Monoclonal Antibody Against Human IgG (Anti-Human IgG)." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 39(5), p. 190FiguresReferencesRelatedDetails Volume 39Issue 5Oct 2020 InformationCopyright 2020, Mary Ann Liebert, Inc., publishersTo cite this article:Ruiyin Chu.Monoclonal Antibody Against Human IgG (Anti-Human IgG).Monoclonal Antibodies in Immunodiagnosis and Immunotherapy.Oct 2020.190-190.http://doi.org/10.1089/mab.2020.0034Published in Volume: 39 Issue 5: October 21, 2020PDF download

Novel Protease-Free Long-Lasting Chemiluminescence System Based on the Dox-ABEI Chimeric Magnetic DNA Hydrogel for Ultrasensitive Immunoassay.

Most of chemiluminescence (CL) substrates exhibit the flash-type light emission. Therefore, the long-lasting CL system is always the crown in the field of CL-based analysis methodology. In this work, we constructed a Dox-ABEI chimeric magnetic DNA hydrogel (MDH) as a novel protease-free long-lasting CL reaction system. The functional MDH can transform flash-type ABEI/H2O2/CO2+ reaction into a glow-type CL system because of its block effect on delaying the diffusion rate of co-reactants, making the CL reaction gradually occur. More importantly, the functional MDH possessed the advantages of biocompatibility and controllability and could be well-designed to incorporate different biosensing strategies. Subsequently, we established a functional MDH-based long-lasting CL immunoassay system for ultrasensitive and highly specific detection of d-dimer and fibrin degradation products (FDPs). The designed CL immunoassay can detect d-dimer and FDP down to 53.7 and 31.6 fg/mL, respectively, with a wide line ranging from 100 fg/mL to 100 ng/mL, which was superior to the previously reported CL biosensing strategies. Moreover, benefiting from the magnetic separation of MDH and excellent CL performance, the developed immunoassaying method was successfully applied in the detection of clinical samples, which showed a close correlation with clinical reference technology. Thus, this functional MDH proved to be an excellent long-lasting CL system and a potential technical platform for clinical bioanalysis applications.

Recent advances in surface-enhanced Raman spectroscopy for the detection of tumor markers

Sensitive and accurate detection of tumor markers is critically essential for the early clinical diagnosis, treatment and prognosis of cancers. Surface-enhanced Raman spectroscopy (SERS) utilizes plasmonic nanomaterials to generate a strong surface plasmon resonance (SPR) effect in the visible to near-infrared region, such as silver and gold nanostructures. The SPR effect can significantly enhance the Raman signals of adsorbed molecules on the surface of nanostructures, allowing to gain the sensitive fingerprint information for the analyte itself or the Raman reporter. Benefitted from the superior properties of high sensitivity, good selectivity, fast and noninvasive analysis, SERS has attracted increasing interest and become a very promising technique for biological analysis. In comparison with other bioanalytical techniques, such as enzyme-linked immunosorbent assay, fluorescence and electrochemical methods, SERS possesses the following advantages: (1) SERS has an ultrahigh sensitivity, even down to single molecule level; (2) SERS can provide the intrinsic molecular fingerprint information, and the bandwidths of SERS peaks are usually very narrow; (3) SERS is suitable for long-term monitoring, because of the resistance of photobleaching and photodegradation; (4) non-invasive detection can be achieved with a low amount of sample and no preparation; (5) the interference of water is very weak, so biological samples can conveniently be detected in aqueous solutions; (6) various SERS-active nanostructures can be designed for multiplex detection. In this review, we provide an overview of recent advances in SERS technology for the detection of different tumor markers, including proteins, enzymes, nucleic acids, cells, tissues, gases and others. Great successes have been achieved by utilizing SERS, which can facilitate the fabrication of assays for tumor biomarkers. However, SERS has not been widely applied in clinical bioanalysis and biodiagnosis in the past over 40 years. SERS-based bioassay involves complex interactions between light, surface plasmon nanomaterials, and bioassay systems. It is difficult to obtain reproducible and reliable Raman spectroscopy data, which is one of the main bottlenecks in SERS bioassay, resulting in the deficiency of clinical and standardized applications. Therefore, there are still many efforts to be done to control the potential factors: (1) It is necessary to prepare a SERS substrate with high uniformity, high enhancement, clean, or/and to properly modify its surface; (2) the suitable Raman reporters and tags are selected to improve the biocompatibility and avoid unspecific adsorption, which can trap analytes in the “hot spots”; (3) biological samples are retained in their native state during the storage and pretreatment procedure to avoid irreversible degradation; (4) appropriate SERS detection conditions are performed, such as excitation wavelength, exposure time, and laser power, to obtain real detection information; (5) accurate biomolecule database are established for spectral assignments. By further improving these challenges, we anticipate the more widespread application of SERS-based assay in biomedicine and bioanalysis fields.

Electrochemical Immunosensors for Sensitive Detection of Neuron-Specific Enolase Based on Small-Size Trimetallic Au@Pd^Pt Nanocubes Functionalized on Ultrathin MnO2 Nanosheets as Signal Labels.

Medically, neuron-specific enolase (NSE) as a specific tumor marker has become an important indicator to diagnose small-cell lung carcinoma. In this study, a sandwich-type electrochemical immunosensor was designed to determine NSE sensitively. Au nanoparticle (Au NP)-embedded zinc-based metal-organic frameworks (Au@MOFs) were prepared as the substrate materials to modify the electrode and immobilize the primary antibody (Ab1). The Au@MOFs with the free amino groups on the MOF surface could effectively increase the immobilization amount of Ab1 through covalent linkage. Simultaneously, the embedding of Au NPs improved the conductivity of MOFs and accelerated interface electron transfer. Sub-30 nm trimetallic Au@Pd^Pt nanocubes (Au@Pd^Pt NCs) loaded onto ultrathin MnO2 nanosheets (MnO2 UNs/Au@Pd^Pt NCs) acted as the labels of secondary antibodies. The small-size Au@Pd^Pt NCs enhanced atomic utilization efficiency and offered more catalytic active sites. The MnO2 UNs with high external surface areas could improve the dispersion of Au@Pd^Pt NCs. The MnO2 UNs/Au@Pd^Pt NCs could catalyze the H2O2 reduction and promote the oxidation of hydroquinone to quinone effectively because of their synergistic effect; thus, the generated quinone achieved amplification of the highly reductive peak current. Furthermore, under the optimal conditions, the immunosensor exhibited a low detection limit (4.17 fg/mL) and broad linear range (10 fg/mL to 100 ng/mL). The results were satisfactory for NSE detection in human serum samples, implying that the presented method had great application potential in clinical bioanalysis.

Cerium oxide-monoclonal antibody bioconjugate for electrochemical immunosensing of HER2 as a breast cancer biomarker

Metal oxide-based sensors have the advantage of rapid response and of high sensitivity to detect specific active biological species and are relatively inexpensive. This report of the present study concerns the development of a cerium oxide – monoclonal antibody bioconjugate for its application as a sensitive immunosensor to detect a breast cancer biomarker. A cerium oxide-anti HER2 bioconjugate was constructed by adding anti HER2 onto cerium oxide that had been previously reacted with APTMS and PEG-NHS-Maleimide. The FTIR spectra of the reaction product showed that the cerium oxide-anti HER2 bioconjugate was successfully synthesized. The resulted bioconjugate was then immobilized on a screen-printed carbon‑gold nanoparticles electrode surface by using the amine coupling bonding systems. The interaction of the synthesized cerium oxide-anti-HER2 bioconjugate with HER2 was found to inhibit an electron transfer and a decrease in the voltammetric Fe(CN)63-/4- peak current, which was proportional to the concentration of HER2. The optimal response of the current signal was generated at an anti-HER2 concentration of 5.0 μg/mL. The two linear ranges of HER2 concentration were found: that were 0.001 to 0.5 ng/mL and 0.5 to 20.0 ng/mL. By using the first calibration curve, the limit of detection was 34.9 pg/mL. The developed label-free immunosensor was used to determine HER2 in a human serum sample with satisfactory results, as shown by a consistent result with the addition of standard. Thus, the resulted immunosensor in this study is promising and has a potential application in clinical bio-analysis.

Thionin functionalized signal amplification label derived dual-mode electrochemical immunoassay for sensitive detection of cardiac troponin I

A sensitive sandwich-type electrochemical immunosensor was established by employing Au@Pt core-shell multi-branched nanoparticles, and thionin functionalized nitrogen/sulfur co-doped graphene oxide (N/S-cGO/L-lys/Au@Pt MBs/Thi) as a double signal label to detect cardiac troponin I (cTnI). In this work, Au nanorods functionalized polydopamine (Au NR@PDA) with high adsorption capacity and superior electroconductivity can provide an efficient substrate for immobilizing primary antibodies (Ab1). In the proposed N/S-cGO/L-lys/Au@Pt MBs/Thi, an electrochemically active molecule, Thi was covalently bonded in the N/S-cGO/L-lys/Au@Pt MBs. It presented a strong differential pulse voltammetry (DPV) current signal without electron transfer mediators, and showed a high electrocatalytic activity toward H2O2 reduction by using amperometric i-t (i-t). Impressively, with the synergistic effect of N/S-cGO/L-lys/Au@Pt MBs/Thi and Au NR@PDA, the developed dual-mode electrochemical immunosensor for cTnI detection showed a wide linear concentration range (50 fg/mL to 250 ng/mL, 750 fg/mL to 100 ng/mL) and a low detection limit (16.7 fg/mL, 250 fg/mL) via i-t and DPV, respectively. Furthermore, this immunosensor exhibited acceptable reproducibility, high sensitivity and good stability under optimal conditions. More importantly, the satisfactory results were obtained in detection of cTnI-spiked human serum samples, and the presented method may be a promising application in clinical bioanalysis.

Multiplex LC-MS/MS Assays for Clinical Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin Analogue Attached via Cleavable Linker to a Biparatopic Humanized Antibody against HER-2.

Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).