Anti-cortisol Antibody Research Articles

Iridium oxide-modified reference screen-printed electrodes for point-of-care portable electrochemical cortisol detection

Cortisol is a well-known stress biomarker; this study focuses on using electrochemical immuno-sensing to measure the concentration of cortisol selectively and sensitively in artificial samples. Anti-cortisol antibodies have been immobilised on polycrystalline Au electrodes via strong covalent thiol bonds, fabricating an electrochemical bio-immunosensor for cortisol detection. IrOx was then anodically electrodeposited as a reference electrode on a commercial screen-printed electrode and electrochemical impedance spectrometry (EIS) studies were used to correlate the electrochemical response to cortisol concentration and the induced changes in charge transfer resistance (Rct). A linear relationship between the Rct and the logarithm of cortisol concentration was found in concentrations ranging from 1 ng/mL to 1 mg/mL with limit of detection at 11.85 pg/mL (32.69 pM). The modification of the reference electrode with iridium oxide has greatly improved the reproducibility of the screen-printed electrode. The sensing system can provide a reliable and sensitive detection approach for cortisol measurements.

Molecular Origins of Long-Term Changes in a Competitive Continuous Biosensor with Single-Molecule Resolution.

Biosensing by particle motion is a biosensing technology that relies on single-molecule interactions and enables the continuous monitoring of analytes from picomolar to micromolar concentration levels. However, during sensor operation, the signals are observed to change gradually. Here, we present a comprehensive methodology to elucidate the molecular origins of long-term changes in a particle motion sensor, focusing on a competitive sensor design under conditions without flow. Experiments were performed wherein only the particles or only the surfaces were aged in order to clarify how each individual component changes over time. Furthermore, distributions of particle motion patterns and switching activity were studied to reveal how particle populations change over timespans of several days. For a cortisol sensor with anticortisol antibodies on the particles and cortisol analogues on the sensing surface, the leading hypotheses for the long-term changes are (i) that the particles lose antibodies and develop nonspecific interactions and (ii) that analogue molecules dissociate from the sensing surface. The developed methodologies and the acquired insights pave a way for realizing sensors that can operate over long timespans.

Ultrasensitive cortisol electrochemical immunosensor amplifying by Au single-atom nanozymes and HRP enzymes

Cortisol, a corticosteroid hormone as a primary stress hormone response to internal and external stress, has been regarded as a gold standard reliable biomarker to evaluate human mental stress. The double enzymes strategy, using nanozyme and enzyme amplifying the electrochemical signal, has been widely used to improve the performance of electrochemical biosensors. An ultra-sensitive electrochemical cortisol sensor based on Au single-atom nanozymes had been fabricated through HRP labeled anti-cortisol antibody binding with Au by Au-S bond. Based on the high catalytic activity of Au single-atom nanozymes and the high selectivity of HRP-labeled anti-cortisol antibodies, the cortisol electrochemical sensor-based Au single-atom nanozymes had an excellent response to cortisol, such as high electrochemical activity, high sensitivity, high selectivity, and wide linear range (0.15–300 ng mL−1) and low detection (0.48 pg mL−1) through the four-parameter logistic model with 95% confidence. The electrochemical cortisol sensor was used to determine the cortisol concentration of human saliva at different times.

Electrochemical Multiplexed N-Terminal Natriuretic Peptide and Cortisol Detection in Human Artificial Saliva: Heart Failure Biomedical Application

The early detection at low concentration, by non-invasive methods, of cardiac biomarkers in physiological fluids has attracted the interest of researchers over the last decade. This enables early diagnosis and prediction of the first signs of heart failure (HF). In this respect, the analysis of human saliva remains the most suitable medium for this non-invasive approach, as it contains a highly interesting biological matrix for general health and disease monitoring. In this work, we developed a highly sensitive multiplexed immunosensor for direct simultaneous detection of both N-terminal Natriuretic Peptide (NT-proBNP) and Cortisol in human artificial saliva (AS). The developed biosensor platform based on silicon nitride substrate was composed from four gold working microelectrodes (WEs) and an integrated counter and reference microelectrode. Gold WEs were biofunctionalized through carboxyl diazonium (4-APA) to immobilize both anti-NT-proBNP and anti-Cortisol antibodies for simultaneous detection. The electroaddressing of the 4-APA onto the gold WE surfaces was realized with cyclic voltammetry (CV), while the interaction between antibodies and antigens in PBS was monitored using electrochemical impedance spectroscopy (EIS). The antigen detection in human AS was realized with EIS combined with the standard addition method. The immunosensor was highly sensitive and selective toward the corresponding biomarkers in both PBS and artificial human saliva as well as in the presence of other potential interfering biomarkers such as tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10). The limit of detection (LOD) was at 0.2 pg/mL for NT-proBNP within the range of 0.03 to 0.9 pg/mL, while the LOD for Cortisol was 0.06 ng/mL within the range of 0.02 to 0.6 ng/mL for Cortisol in artificial saliva. The developed immunosensor is very promising for significant detection in physiological media, and time reducing as it allows the simultaneous detection of various biomarkers.

Structural insight to elucidate the binding specificity of the anti-cortisol Fab fragment with glucocorticoids

Cortisol is a steroid hormone that is produced by the adrenal gland. It is a primary stress hormone that increases glucose levels in the blood stream. High concentrations of cortisol in the body can be used as a biomarker for acute and chronic stress and related mental and physiological disorders. Therefore, the accurate quantification of cortisol levels in body fluids is essential for clinical diagnosis. In this article, we describe the isolation of recombinant anti-cortisol antibodies with high affinity for cortisol and discover their cross-reactivity with other glucocorticoids. To describe the cortisol binding site and elucidate the structural basis for the binding specificity, the high-resolution crystal structures of the anti-cortisol (17) Fab fragment in the absence of glucocorticoid (2.00 Å) and the presence of cortisol (2.26 Å), corticosterone (1.86 Å), cortisone (1.85 Å) and prednisolone (2.00 Å) were determined. To our knowledge, this is the first determined crystal structure of a cortisol-specific antibody. The recognition of cortisol is driven by hydrophobic interactions and hydrogen bonding at the protein–ligand interface coupled with a conformational transition. Comparison of ligand-free and ligand-bound structures showed that the side chains of residues Tyr58-H and Arg56-H can undergo local conformational changes at the binding site, most likely prior to the binding event via a conformational selection mechanism. Compared to other anti-steroid antibody–antigen complexes, (17) Fab possesses a structurally unique steroid binding site, as the H3 loop from the CDR area has only a minor contribution, but framework residues have a prominent contribution to hapten binding.

Sweat cortisol determination utilizing MXene and multi-walled carbon nanotube nanocomposite functionalized immunosensor

In this work, a highly selective and sensitive cortisol immunosensor based on MXene and multi-walled carbon nanotubes (MXene-MWCNTs) hybrid composite was proposed. The immunosensor was developed by screen printing technique on a flexible polyethyleneterephthalate (PET) substrate followed by modification of MXene-MWCNTs and anti-cortisol antibodies with EDC/NHS coupling agent. Owing to the large surface area and excellent conductivity of MXene-MWCNTs, the immunosensor exhibited a low detection limit and a wide linear range toward cortisol from 0.1 fg/mL to 1 µg/mL, which covered the normal range of free cortisol in sweat. Additionally, the immunosensor was applied to evaluate the cortisol level in human sweat samples, and the results were validated by a commercial chemiluminescence immunoassay (CLIA) assay. The present platform provides an effective method for non-invasive monitoring of human sweat cortisol levels, which may be well-suited for hormone screenings in healthy monitoring and medical diagnosis.

Electrochemical Sensing of Cortisol in Human Saliva and Serum Using DNA-Steroid Conjugation with a Versatile DNA Nanostructure Sensor

Having a generalizable point of care (POC) method for clinically relevant biomolecules would greatly enhance healthcare management and disease diagnosis. Recently our group developed a novel DNA nanostructure architecture for versatile detection of analytes which is generalizable for assaying several small molecules and their larger protein binding partners (e.g. antibodies) in human serum. The DNA nanostructure is built through on-electrode enzymatic ligation of three oligos for electrode attachment, anchor binding, and electrochemical signaling. In this study, we developed an economical synthetic approach for making oligonucleotide-steroid conjugates, and we explored the capability of these DNA nanostructure sensors for small molecule/steroid detection.Cortisol is a steroid hormone secreted by the hypothalamic-pituitary-adrenal system in response to the body’s stress level. As the main stress hormone, it controls processes such as immune, adrenal, circulatory, and metabolic. Moreover, anomalies of cortisol levels can result in serious conditions such as Addison’s disease, Cushing’s syndrome, and adrenal insufficiencies. Every year, 120,000 deaths are attributed, in part, to elevated levels of stress. To minimize these issues, a simplified method for cortisol sensing is vital. In this study, we conjugated cortisol to amine-tagged DNA and used the conjugates in our sensors. Anti-cortisol antibodies induced a 67% signal drop, validating conjugation. A calibration curve for cortisol showed a limit of detection of 800 pM and a dynamic range of 1 – 100 nM. The sensor was validated in saliva and human serum samples using the gold standard method, ELISA. Changing cortisol levels in two human patients' saliva samples were successfully detected after several collection times throughout a single day. As a potential point-of-care (POC) detection device, our novel cortisol biosensor could detect the hormone in human serum and saliva samples within 6 minutes.

Immuno field-effect transistor (ImmunoFET) for detection of salivary cortisol using potentiometric and impedance spectroscopy for monitoring heart failure

Cortisol, a steroid hormone mostly known as “the stress hormone,” plays many essential functions in humans due its involvement in several metabolic pathways. It is well-known that cortisol dysregulation is implied in evolution and progression of several chronic pathologies, including cardiac diseases such as heart failure (HF). However, although several sensors have been proposed to date for the determination of cortisol, none of them has been designed for its determination in saliva in order to monitor HF progression. In this work, a silicon nitride based Immuno field-effect transistor (ImmunoFET) has been proposed to quantify salivary cortisol for HF monitoring. Sensitive biological element was represented by anti-cortisol antibody bound onto the ISFET gate via 11-triethoxysilyl undecanal (TESUD) by vapor-phase method. Potentiometric and electrochemical impedance spectroscopy (EIS) measurements were carried out for preliminary investigations on device responsiveness. Subsequently, a more sensitive detection was obtained using electrochemical EIS. The proposed device has proven to have a linear response (R2 always >0.99), to be sensitive (with a limit of detection, LoD, of 0.005 ± 0.002 ng/mL), selective in case of other HF biomarkers (e.g. N-terminal pro B-type natriuretic peptide (NT-proBNP), tumor necrosis factor-alpha (TNF-α), and interleukin 10 (IL-10)), and accurate in cortisol quantification in saliva sample by performing the standard addition method.

Signal-on photoelectrochemical immunoassay for salivary cortisol based on silver nanoclusters-triggered ion-exchange reaction with CdS quantum dots.

Nowadays, the epidemic, employment, and academic pressures are seriously affecting our physical and mental health. Herein, we designed a magneto-controlled photoelectrochemical immunosensor for noninvasive monitoring of salivary cortisol regarded as a pressure biomarker. A competitive immunoassay model was established by coupling bovine serum albumin-cortisol modified magnetic beads (MB-BSA-cortisol) with silver nanoclusters (Ag NCs)-labelled anti-cortisol antibody, and quantity analysis was operated by photoelectrochemical measurement of the CdS/Au electrode as an ion-exchange platform. Accompanying the formation of immune complexes, the carried Ag NCs were readily dissolved with nitric acid to produce abundant silver ions, which transferred to the electrode for ion-exchange reaction with CdS quantum dots to produce Ag2S, a new electron–hole capture site, leading to a decrease in the photocurrent intensity. The photocurrent signal gradually recovered with the increase of concentration of target cortisol, acquiring the signal-on mode competitive immunosensing system, which is propitious to the detection of small molecules. Within optimal conditions, this sensor had a satisfactory linear relationship in the range of 0.0001–100 ng mL−1 with favorable repeatability, specificity, and acceptable method accuracy. The detection limit was as low as 0.06 pg mL−1. In addition, this strategy provided new thought for the test of other small-molecule analytes and immunosensor applied in the complex biological system.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-022-03893-z.

Portable Chemiluminescence-Based Lateral Flow Assay Platform for the Detection of Cortisol in Human Serum.

In this study, we developed the portable chemiluminescence (CL)-based lateral flow assay (LFA) platform for the detection of cortisol in human serum. Cortisol is well-known as a stress hormone due to its high relevancy for human mental and physical health, such as hypertension or depression. To date, a number of optical devices have provided the sensitive determination of levels of analytes. However, this modality type still requires costly optical modules. The developed CL platform is simply composed of two detection modules along with a loading part for the LFA strip. The LFA membrane contains gold nanoparticle probes conjugated with antibodies against cortisol and horseradish peroxidase (HRP), which can also efficiently increase the luminescent signal by providing many areas for anti-cortisol antibody and HRP. The measured voltage signals coming from the photodiode in a CL reader were compared with a standard microplate reader for the evaluation of accuracy. The linear range observed for cortisol was measured to be 0.78–12.5 μg/dL (R2 = 0.99) with a limit of detection (LOD) of 0.342 μg/dL. In addition, the CL-LFA reader showed a high correlation (R2 = 0.96) with the standard cortisol console (COBAS 8000, Roche), suggesting that our developed CL-based LFA platform can be usable in situ.

Cortisol AuPd plasmonic unclad POF biosensor

This paper presents the development and feasibility tests of a cortisol immunosensor. The sensor is based on surface plasmon resonance (SPR) using an unclad plastic optical fiber (POF) in which the SPR is used as sensitivity enhancer, promoted by a gold/palladium (AuPd) alloy coating. The AuPd coated fibers were functionalized with an anti-cortisol antibody and passivated with bovine serum albumin (BSA) to be tested in the presence of cortisol as target analyte. The antibody-antigen binding reaction caused a variation of the refractive index on the surface of the AuPd coating, which leads to a shift of the SPR signature wavelength. The sensor was tested for different cortisol concentrations, ranging from 0.005 to 10 ng/mL. The reported biosensor presented a total wavelength shift of 15 nm for the testing range, putting in evidence a high sensitivity. Control tests for selectivity assessment were also performed. Concentrations as high as 10 ng/mL of cortisol, in a sensor functionalized with anti-hCG antibodies, only resulted in 1 nm variation of the resonance wavelength, 15 times lower than the one functionalized with the anti-cortisol antibodies, which indicates a high selectivity for the proposed approach. For this sensing approach the limit of detection (LOD) was determined to be 1 pg/mL. The proposed SPR based POF sensor has a low-cost interrogation method, high sensitivity and low LOD, straightforward signal processing and find important applications in different biological fields.

Anxiety determination by antibody-conjugated nanoparticles on an interdigitated electrode sensor.

This work focused on the detection of cortisol on an interdigitated electrode sensor surface using an anti-cortisol antibody. To improve immobilization, antibodies were conjugated with silver nanoparticles and attached to the surface of the sensor. Cortisol interacted in a dose-dependent manner on the antibody-immobilized sensor surface, and current changes were observed. Linear regression analysis was performed by a 3σ calculation, and the limit of detection fell into the range of 0.01 and 0.1ng/mL. The sensitivity of cortisol was calculated to be 0.01ng/mL and the sensor discriminated against other hormones, namely norepinephrine and progesterone, with higher selectivity for cortisol. This result represented the selective detection of cortisol with high performance, which can help to determine anxiety disorders.

Label-Free Sensitive Detection of Steroid Hormone Cortisol Based on Target-Induced Fluorescence Quenching of Quantum Dots.

We discovered that several types of steroid hormones quench the fluorescence of quantum dots (QDs) at close proximity. Inspired by the finding, we developed a new type of biosensor for the sensitive detection of cortisol via direct fluorescence quenching of functionalized QD probes directly induced by the capture of target cortisol without additional reporter reagents. The detection selectivity was provided by cortisol-selective aptamers or anticortisol antibodies conjugated on the QD surfaces. With the magnetic nanoparticle labeling, the new sensing method enabled rapid cortisol sensing at physiologically relevant concentrations and yielded the detection limit of ∼1 nM for aptamer-based sensors and ∼100 pM for antibody-based sensors. We also evaluated the new detection method using saliva samples with an optimized sample preparation process under the assistance of magnetic manipulation. The result showed a satisfying recovery rate for spiked saliva tests. The facile sensing technology offers an appealing approach for the detection of steroid hormones in point-of-care settings.

Cortisol in-fiber ultrasensitive plasmonic immunosensing

Cortisol is a stress biomarker whose chronic elevated levels are associated with higher risk of metabolic syndromes, anxiety, and cardiovascular diseases, among other medical conditions. A new immunosensor based on plasmonic tilted fiber Bragg grating (TFBG) has been developed and tested for rapid and ultrasensitive cortisol detection. The gold coated TFBG was characterized to surrounding refractive index (SRI) changes and functionalized with anti-cortisol antibodies via cysteamine. The functionalization was monitored, allowing to verify the SRI alteration at the fiber surface by the respective molecular adhesion. In this work, an alternative method to the monitoring of the most sensitive surface plasmon resonance mode was explored, based on tracking the local maximum of the plasmonic signature of the lower envelope of the spectra. With this interrogation method, the sensor achieved a sensitivity to cortisol detection of 0.275 ± 0.028 nm/ng.mL−1, for the detection range of 0.1-10 ng/mL, with a total wavelength shift of around 3 nm, which is higher several orders of magnitude than the usually reported TFBG plasmonic immunosensors. The proposed biosensor provides a rapid, highly sensitive, label-free, low-volume consumption method for cortisol detection, with a working range suitable to monitor different biological samples.

In vitro affinity maturation of anti-cortisol antibodies to develop sensitive immunoassays

In vitro affinity maturation of anti-cortisol antibodies to develop sensitive immunoassays

Electrochemical-digital immunosensor with enhanced sensitivity for detecting human salivary glucocorticoid hormone.

In this work, an ultra-sensitive electrochemical-digital sensor chip is devised for potential use as a digital stress analyzer for point-of-care testing (POCT) and preventive on-site recording of the hormone 'cortisol', a glucocorticoid class of steroid hormone present in the human saliva. The sensor was interfaced and re-configured with a high precision impedance converter system (AD5933) and used for electrochemical impedance spectroscopy (EIS) to evaluate the cortisol levels in seven saliva samples. To obtain enhanced biological (cortisol) recognition and achieve a lower limit of detection 0.87 ± 0.12 pg mL-1 (2.4 ± 0.38 pmol mL-1) with a wide range from 1 pg mL-1 to 10 ng mL-1 (2.75 pmol mL-1 to 27.58 pmol mL-1; R2 = 0.9831), bovine serum albumin (1% BSA) was utilized as an effective sensitivity enhancer in addition to optimizing the other two parameters: (i) anti-cortisol antibody (anti-CAb) covalently attached to micro-Au electrodes and (ii) saliva sample incubation time on the sensor chip. The results obtained in this work were corroborated with the gold standard ELISA test with an accuracy of 96.3% and other previously reported biosensors. We envisage that the conceivable standpoint of this study can be a practice towards new development in cortisol biosensing, which will be pertinent to POCT targeted for in vitro psychobiological study on patient cortisol in saliva, and finally an implantable sensor chip in the future.

High-Contrast Facile Imaging with Target-Directing Fluorescent Molecular Rotors, the N3-Modified Thioflavin T Derivatives.

Immunostaining methods have generally been used not only for biological studies but also for clinical diagnoses for decades. However, recently, for these methods, improved rapidity and simplicity have been required for relevant techniques in laboratory research and medical applications. To this end, we present here a novel approach for designing fluorescent molecular rotor probes, i.e., N3-modified thioflavin T (ThT) derivatives, which enabled specific detection of interesting protein targets with sensitive fluorescence turn-on. As an example, we synthesized N3-( d-desthiobiotinyl-PEGylated) thioflavin T (ThT-PD) and N3-(cortisolyl-PEGylated) thioflavin T (ThT-PC) that carried d-desthiobioin and cortisol, respectively, via PEG linkers. Compared to those of the probes without the targets, ThT-PD and ThT-PC exhibited around 27- and 8-fold fluorescence intensities, respectively, with the target streptavidin and anti-cortisol antibody in excess of saturation, enabling quantitative detection of the targets. Furthermore, we successfully demonstrated the feasibility of ligand-tethering N3-ThT derivatives by the rapid specific staining of glucocorticoid receptors in cells, which was completed within only several minutes using ThT-PC after cell fixation, whereas it took ∼24 h for immunostaining to capture the corresponding fluorescence images.

Local preparation and evaluation of liquid phase radioimmunoassay for determination of human serum cortisol

The main objective of the present study was the preparation of the primary reagents of cortisol radioimmunoassay (RIA) using liquid phase double antibody technique. Three basic components were prepared and characterized to obtain valid and accurate system. These components were polyclonal anti-cortisol antibody, 125I-cortisol RIA tracer and cortisol standards. Cortisol requires conjugation step to be real antigen. Therefore, in this study this mandatory conjugation step was performed & characterized. Then formulation, optimization and validation of this liquid phase RIA technique were carried out. This assay is precise, specific and sensitive enough for using as a diagnostic tool for the adrenal cortex status.

Paper-Based Analytical Biosensor Chip Designed from Graphene-Nanoplatelet-Amphiphilic-diblock-co-Polymer Composite for Cortisol Detection in Human Saliva.

Cortisol has been identified as a biomarker in saliva to monitor psychological stress. In this work, we report a label-free paper-based electrical biosensor chip to quantify salivary cortisol at a point-of-care (POC) level. A high specificity of the sensor chip to detect cortisol with a detection limit of 3 pg/mL was achieved by conjugating anticortisol antibody (anti-CAB) on top of gold (Au) microelectrodes using 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester (DTSP) as a self-assembled monolayer (SAM) agent. The electrode design utilized poly(styrene)-block-poly(acrylic acid) (PS67-b-PAA27) polymer and graphene nanoplatelets (GP) suspension coated on filter paper to increase the sensitivity of the immune response. A biosensor chip was then integrated with a lab-built low-cost miniaturized printed circuit board (PCB) to provide an electrical connection and to wirelessly transmit/receive electrical signals using MATLAB. This fully integrated proposed hand-held device successfully exhibited a wide cortisol-detection range from 3 pg/mL to 10 μg/mL, with a sensitivity of 50 Ω (pg mL-1)-1. The performance of the proposed cortisol sensor chip was validated using an enzyme-linked immunosorbent assay (ELISA) technique with a regression value of 0.9951. The advantages of the newly developed cortisol immune biosensor over previously reported chips include an improved limit of detection, no need for additional redox medium for electron exchange, faster response to achieve stable data, excellent shelf life, and its economical production.

A Single-Step "Breeding" Generated a Diagnostic Anti-cortisol Antibody Fragment with Over 30-Fold Enhanced Affinity.

Cortisol levels in bodily fluids represent a useful index for pituitary-adrenal function, and thus practical anti-cortisol antibodies are required. We have studied "antibody-breeding" approaches, which involve in vitro evolution of antibodies to improve their antigen-binding performances. Here, we produced an antibody fragment to measure serum cortisol levels with over 30-fold enhanced affinity after single mutagenesis and selection steps. A mouse anti-cortisol antibody, Ab-CS#3, with insufficient affinity for practical use, was chosen as the prototype antibody. A "wild-type" single-chain Fv fragment (wt-scFv; Ka, 3.4×108 M-1) was prepared by bacterial expression of a fusion gene combining the VH and VL genes for this antibody. Then, random point mutations were generated separately in VH or VL by error-prone PCR, and the resulting products were used to assemble scFv genes, which were displayed on filamentous phages. Repeated panning of the phage library identified a mutant scFv (scFv#m1-L10) with an over 30-fold enhanced affinity (Ka 1.2×1010 M-1). Three amino acid substitutions (Cys49Ser, Leu54Pro, and Ser63Gly) were observed in its VL sequence. In a competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with measuring range ca. 0.03-0.6 ng/assay cortisol, midpoint of which (0.15 ng/assay) was 7.3-fold lower than that of wt-scFv. Although cortisone, 11-deoxycortisol, and prednisolone showed considerable cross-reactivity, the mutant scFv should enable sensitive routine cortisol assays, except for measurement after metyrapone or high-dose of prednisolone administrations. Actually, cortisol levels of control sera obtained with the scFv-based ELISA were in the reference range.