Optical Immunoassay Research Articles

Optical fiber SPR probe platform combined with oriented antibody optimized modification for ultrasensitive and portable detection of human thyroglobulin

Thyroglobulin (Tg) is an essential indicator for monitoring differentiated thyroid cancer. However, there are still significant challenges in achieving fast, accurate, and efficient point-of-care testing (POCT). This paper presented an optical fiber bioprobe platform technology based on the ultrasensitive sensing principle of surface plasmon resonance (SPR), aiming to achieve an efficient and precise immunoassay of Tg. The fiber probes were modified with Tg antibodies to form fiber Tg probes. After optimization of the oriented antibody modification, the detection sensitivity of fiber Tg probes increased by 3 times, facilitating one-step direct detection of μg-level sample concentrations such as thyroid tissue fluid samples. For ng-level sample concentrations such as blood samples, a two-step measurement method was used to amplify the surface load signal. Through Tg detection studies in different concentration ranges and background environments, our fiber Tg probes exhibited a sensitivity of 81.99 pm / (ng / mL) and a limit of detection of 1.4 ng / mL, and were able to complete ng-level detection in serum backgrounds. The fiber Tg probe holds immense potential in the diagnosis and postoperative monitoring of thyroid cancer. Additionally, being a miniature, disposable, reliable, and portable quantitative optical immunoassay tool, it can serve as a rapid detection instrument for various target proteins and pathogens by simply altering the modified antibody.

Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: Collaborative Study, Final Action 2021.07.

Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of growth, immunoprotective, and antimicrobial factors to the neonate. To evaluate method reproducibility of AOAC First Action Official Method 2021.07 for compliance with the performance requirements described in Standard Method Performance Requirement (SMPR®) 2020.005. Eight laboratories participated in the analysis of blind-duplicate samples of seven nutritional products. Samples were diluted in buffer, and an optical biosensor immunoassay was used in a direct-assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a four-parameter calibration regression. After outliers were removed, precision as reproducibility was found to be within limits set in SMPR 2020.005 (≤ 9%) for six out of seven samples and all had acceptable Horwitz Ratio (HorRatR) values ranging from 1.0 to 2.1. Additionally, comparison with an alternative independent Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) First Action method (heparin cleanup LC-UV), showed negligible difference between results. The method described is suitable for the quantification of intact, undenatured bovine lactoferrin in powdered infant formulas. The SPIFAN Expert Review Panel evaluated the method and accompanying validation data from this multi-laboratory testing (MLT) study in July 2023 and recommended Official Method 2021.07 for adoption as a Final Action Official MethodSM. A multi-laboratory validation study of an automated optical biosensor immunoassay for the determination of intact, undenatured bovine lactoferrin is described.

Realization of high-performance biosensor through sandwich analysis utilizing weak value amplification

A label-free optical sandwich immunoassay sensor, utilizing weak value amplification and total internal reflection, was devised for real-time, high-sensitivity analysis and detection of low-concentration targets. 3D printed channels and sodium chloride solution were employed to ensure reproducibility, reliability, and stability of the measurements for calibration. The sandwich structure demonstrated enhanced responsiveness in the proposed optical biosensor through a comparative analysis of the direct assay and sandwich assay for detecting alpha-fetoprotein (AFP) at the same concentration. By optimizing the binding sequences of the coating antibody, target, and detection antibody in the sandwich method, a more suitable sandwich sensing approach based on weak value amplification was achieved. With this approach, the limit of detection (LOD) of 6.29 ng/mL (pM level) for AFP in PBS solution was achieved. AFP testing and regeneration experiments in human serum have proved the feasibility of our methods in detecting complex samples and the reusability of sensing chips. Additionally, the method demonstrated excellent selectivity for unpaired antigens. The efficacy of this methodology was evaluated by simultaneously detecting AFP, carcinoembryonic antigen (CEA), and CA15-3 on a singular sensor chip. In conclusion, the label-free sandwich immunoassay sensing scheme holds promise for advancing the proposed optical sensors based on weak value amplification in early diagnosis and prevention applications. Compared to other biomarker detection methods, it will be easier to promote in practical applications.

A colorimetric immunoassay for the detection of human vascular endothelial growth factor 165 (VEGF165) based on anti-VEGF-iron oxide nanoparticle conjugation

Vascular endothelial growth factor (VEGF) is an indispensable element in many physiological processes, while alterations in its level in the circulating system are signs of pathology-associated diseases. Therefore, its precise and selective detection is critical for clinical applications to monitor the progression of the pathology. In this study, an optical immunoassay biosensor was developed as a model study for detecting recombinant VEGF165. The VEGF165 sample was purified from recombinant Kluyveromyces lactis GG799 yeast cells. Indirect ELISA was used during the detection, wherein iron oxide nanoparticles (FeNPs) were utilized to obtain optical signals. The FeNPs were synthesized in the presence of lactose p-amino benzoic acid (LpAB). VEGF165 antibody was conjugated to the LpAB-FeNPs through EDC/NHS chemistry to convert the iron oxide nanoparticles into VEGF165 specific probes. The specificity of the prepared system was tested in the presence of potential serum-based interferents (i.e., glucose, urea, insulin, C-reactive protein, and serum amyloid A), and validation studies were performed in a simulated serum sample. The proposed immunoassay showed a wide detection range (0.5 to 100 ng/mL) with a detection limit of 0.29 ng/mL. These results show that the developed assay could offer a sensitive, simple, specific, reliable, and high-throughput detection platform that can be used in the clinical diagnostics of VEGF.Graphical

Zinc Alpha-2 Glycoprotein, Acylated Ghrelin, and Zinc Levels in Prediabetics.

Prediabetic stages of impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) exhibit differences in the sites of insulin resistance. Serum Zinc α-2 glycoprotein (ZAG), acylated ghrelin (AG), and zinc (Zn) levels can affect IFG, IGT, and diabetic glucose tolerance (DGT) differently. This study examined the importance of ZAG, AG, and serum Zn levels in prediabetic individuals with IFG, IGT, and DGT, compared to those with normal glucose levels. The study was conducted at İstanbul University Cerrahpaşa-Cerrahpaşa Faculty of Medicine. A total of n=151 volunteers were classified according to the WHO criteria for diabetes after undergoing an oral glucose tolerance test. Plasma and serum samples were measured by Inductively Coupled Plasma Optical Emission Spectroscopy, ELISA, and immunoassay. Prediabetic conditions became more prominent with the decrease in ZAG levels. ZAG levels showed a negative correlation with acylated ghrelin and Homeostatic Model Assessment for assessing beta-cell function and insulin resistance. Zinc levels were significantly lower in DGT. ZAG levels have regulatory effects on insulin resistance and plasma glucose levels are mediated by zinc and acylated ghrelin.

Temporal Profiles of P-Tau, T-Tau, and P-Tau:Tau Ratios in Cerebrospinal Fluid and Blood from Moderate-Severe Traumatic Brain Injury Patients and Relationship to 6-12 Month Global Outcomes.

Traumatic brain injury (TBI) can initiate progressive injury responses, which are linked to increased risk of neurodegenerative diseases known as "tauopathies." Increased post-TBI tau hyperphosphorylation has been reported in brain tissue and biofluids. Acute-to-chronic TBI total (T)-tau and phosphorylated (P)-tau temporal profiles in the cerebrospinal fluid (CSF) and serum and their relationship to global outcome is unknown. Our multi-site longitudinal study examines these concurrent profiles acutely (CSF and serum) and also characterizes the acute- to-chronic serum patterns. Serial serum and CSF samples from individuals with moderate-to-severe TBI were obtained from two cohorts (acute, subacute, and chronic samples from University of Pittsburgh [UPitt] [n = 286 unique subjects] and acute samples from Baylor College of Medicine [BCM] [n = 114 unique subjects]) and assayed for T-tau and P-tau using the Rolling Circle Amplification-Surround Optical Fiber ImmunoAssay platform. Biokinetic analyses described serum T-tau and P-tau temporal patterns. T-tau and P-tau levels are compared with those in healthy controls (n = 89 for both CSF and serum), and univariate/multivariable associations are made with global outcome, including the Disability Rating Scale (DRS) and the Glasgow Outcome Scale-Extended (GOS-E) scores at 3 and 6 months post-TBI (BCM cohort) and at 6 and 12 months post-TBI (UPitt cohort). For both the UPitt and BCM cohorts, temporal increases in median serum and CSF T-tau and P-tau levels occurred over the first 5 days post-injury, while the initial increases of P-tau:T-tau ratio plateaued by day 4 post-injury (UPitt: n = 99, BCM: n = 48). Biokinetic analyses with UPitt data showed novel findings that T-tau (n = 74) and P-tau (n = 87) reached delayed maximum levels at 4.5 and 5.1 days, while exhibiting long serum half-lives (152 and 123 days), respectively. The post-TBI rise in acute (days 2-6) serum P-tau (up to 276-fold) far outpaced that of T-tau (7.3-fold), leading to a P-tau:T-tau increase of up to 267-fold, suggesting a shift toward tau hyperphosphorylation. BCM analyses showed that days 0-6 mean CSF T-tau and P-tau levels and P-tau:T-tau ratios were associated with greater disability (DRS) (n = 48) and worse global outcome (GOS-E) (n = 48) 6 months post-injury. Days 0-6 mean serum T-tau, P-tau, and P-tau:T-tau ratio were not associated with outcome in either cohort (UPitt: n = 145 [DRS], n = 154 [GOS-E], BCM: n = 99 [DRS and GOS-E]). UPitt multivariate models showed that higher chronic (months 1-6) mean P-tau levels and P-tau:T-tau ratio, but not T-tau levels, are associated with greater disability (DRS: n = 119) and worse global outcomes (GOS-E: n = 117) 12 months post-injury. This work shows the potential importance of monitoring post-TBI T-tau and P-tau levels over time. This multi-site longitudinal study features concurrent acute TBI T-tau and P-tau profiles in CSF and serum, and also characterizes acute-to-chronic serum profiles. Longitudinal profiles, along with no temporal concordance between trajectory groups over time, imply a sustained post-TBI shift in tau phosphorylation dynamics that may favor tauopathy development chronically.

Label-free optical and electrical immunoassays based on lyotropic chromonic liquid crystals: Implications of real-time detection and kinetic analysis

Conventional liquid crystal (LC)-based biosensors utilize predominantly thermotropic LCs as the signal-transducing media, which are less environmentally sustainable compared with lyotropic counterparts. In this study, the nematic phase of the anionic azo dye sunset yellow (SSY), a type of lyotropic chromonic liquid crystals (LCLCs), was employed in the optical and electrical biosensing of bovine serum albumin (BSA) and the cancer biomarker CA125. The optical response observed under a polarizing optical microscope was quantified by image analysis, taking advantage of the specific absorption of SSY. The electrical response derived from the dielectric spectra of SSY provided a new alternative for quantitative bioassay based on nematic LCLCs. The limit of detection (LOD) of the optical and electrical protein assay was ∼10−11- and ∼10−10-g/ml BSA, respectively, whereas that of the optical and electrical immunoassay was 5.97 × 10−11 and 6.02 × 10−12 g/ml for CA125, respectively. Moreover, real-time monitoring and kinetic analysis, which are hardly achievable for the hydrophobic thermotropic LCs, were demonstrated by dispersing CA125 in nematic SSY and subsequently recording the optical response over time during the specific binding between CA125 and the immobilized anti-CA125 antibody. Results from this study further the potential of nematic LCLCs in biosensing, especially in dielectric and real-time detection.

Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: First Action 2021.07.

Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of immunoprotective, growth, and antimicrobial factors to the neonate. To evaluate the analytical performance of an optical biosensor immunoassay for compliance with the method performance requirements described in SMPR 2020.005. Following dilution of the sample in buffer, an automated, label-free, real-time optical biosensor immunoassay was used in a direct assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a 4-parameter calibration regression. The analytical range (0-200 mg/hg), method detection limit (0.8mg/hg), recovery (96.1-109.2%), and repeatability (1.0-5.3%) complied with the requirements given in the lactoferrin SMPR. The method was shown to be specific for native, intact lactoferrin; thermally denatured lactoferrin generated no measurable binding response. The method described is suitable for the quantification of intact, undenatured lactoferrin in milk products, infant formulas (bovine milk protein-based, soy protein-based, and amino acid-based), and adult nutritionals and has been demonstrated to meet the performance requirements defined in SMPR 2020.005. A single-laboratory validation (SLV) of an automated biosensor immunoassay for the determination of intact, undenatured lactoferrin is described.

Development of an optical immunoassay based on peroxidase-mimicking Prussian blue nanoparticles and a label-free electrochemical immunosensor for accurate and sensitive quantification of milk species adulteration.

In contrast to reported enzyme-based immunoassays, an enzyme-free immunoassay (optical and electrochemical) is presented here for the first timethat can be used as point-of-need detection bioplatforms of bovine IgG as goat milk adulterant. In the first format, Prussian blue nanoparticles (PBNPs) were used as antibody catalytic labels in a competitive colorimetric microplate immunoassay. Absorbance measurement was performed photometrically at 450nm. After in-depth optimization, excellent sensitivity was achieved (0.01% cow/goatvolume ratio), which is 100 times lower than the limit allowed by the European legislation (EL) (1% v/v), thanks to the high catalytic activity of PBNPs compared withnatural peroxidase. Moreover, the antibody-PBNPs bioconjugates showed excellent stability over 4weeks (> 94% of the initial response) confirming the successful anchoring of the antibodies to the surface of the PBNPs. On the other hand, a label-free voltammetric immunoassay for the detection of bovine IgG was developed. The sensing principle was based on the hindrance of charge transfer between ferri-ferrocyanide redox couple and the screen-printed gold electrodes modified with bovine IgG antibody. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the step-by-step modification of the electrode surface. Under optimal conditions, this single-step electrochemical analysis achieved a high sensitivity of 0.1% (cow/goat) when monitoring the ferrocyanide oxidation at + 0.092V (vs. Ag/AgCl) using differential pulse voltammetry (DPV). The selectivityof the developed immunoassays was evaluated for different species of milk of similar composition, and both immunoassays exhibited a selective response only to bovine IgG. Unlike conventional immunoassays, the developed enzyme-free immunoassays have many attractive features for the detection of milk adulteration, whether they are usedin quality control laboratories for routine milk analysis (optical immunoassay) or at on-site checkpoints (electrochemical immunoassay) using wireless electrochemical detectors. Thesensors providehigh sensitivity (≤ 0.1%), excellent precision (RSD < 6%), low cost (no enzyme is required) and ease of operation, including handling of milk samples.

Quantum optical immunoassay: upconversion nanoparticle-based neutralizing assay for COVID-19

In a viral pandemic, a few important tests are required for successful containment of the virus and reduction in severity of the infection. Among those tests, a test for the neutralizing ability of an antibody is crucial for assessment of population immunity gained through vaccination, and to test therapeutic value of antibodies made to counter the infections. Here, we report a sensitive technique to detect the relative neutralizing strength of various antibodies against the SARS-CoV-2 virus. We used bright, photostable, background-free, fluorescent upconversion nanoparticles conjugated with SARS-CoV-2 receptor binding domain as a phantom virion. A glass bottom plate coated with angiotensin-converting enzyme 2 (ACE-2) protein imitates the target cells. When no neutralizing IgG antibody was present in the sample, the particles would bind to the ACE-2 with high affinity. In contrast, a neutralizing antibody can prevent particle attachment to the ACE-2-coated substrate. A prototype system consisting of a custom-made confocal microscope was used to quantify particle attachment to the substrate. The sensitivity of this assay can reach 4.0 ng/ml and the dynamic range is from 1.0 ng/ml to 3.2 upmug/ml. This is to be compared to 19 ng/ml sensitivity of commercially available kits.

Algorithms for calculating polarization direction based on spatial modulation of vector optical field

Polarization is an important property of electromagnetic waves, and measuring their polarization properties fast and precisely is a very important issue in many applications, such as skylight polarization navigation, optical activity measurement, imaging polarimetry, spectroscopic ellipsometry, and fluorescence polarization immunoassay . The polarization measurement method based on vector optical field modulation and image processing is a new type of spatial modulation polarization detection technology. The key step of this technique moving to practical application is determined by effective polarization measuring algorithms with high speed and accuracy. In order to find out the method of fast and precisely calculating polarization direction, the principle of polarization direction measurement based on vector optical field and spatial modulation is introduced briefly, and the basic characteristics of the spatially modulated intensity distribution images are analyzed. According to the properties of spatially modulated image, we propose and implement four different polarization direction calculation methods, which are the Radon transform, intensity modulation curve detection, radial integration, and image correlation detection, and also introduce their working principles and physical thoughts elaborately. To compare the detailed performances of these four algorithms, an experimental setup is constructed to collect the images and perform the algorithm verification, and the stabilities, speeds and accuracies of the four algorithms are compared. The research results indicate that all the four methods can achieve their stable and reliable polarization direction detections. The three methods, intensity modulation curve detection, radial integration and image correlation detection, can achieve the polarization direction measuring accuracy better than 0.01° . The intensity modulation curve detection and radial integration have relatively fast calculation speed and the best comprehensive performances , and are the most promising methods to realize real-time and high-precision polarization measurement.

A label-free optical immunoassay based on birefringence of liquid crystal for insulin-like growth factor-I sensing

Human insulin-like growth factor-I (IGF-I) is a pleiotropic polypeptide hormone, which has been widely utilized for the diagnosis and management of growth hormone (GH) related disorders and diseases. In this work, we verify a label-free optical biosensor based on nematic liquid crystals (LCs) of 4-cyano-4-pentylbiphenyl (5CB) for ultrasensitive sensing of trace IGF-I in human serum samples. 5CB shows unique birefringence driven by the reorientation of the self-assembled N, N-dimethyl-N-(3-(trimethoxysilyl)propyl)−1-octadecanaminiuchloride (DMOAP) on glass. IGF-I antibody (AbIGF-I), as a specific recognition element, can be immobilized onto modified glass surface through a cross-linker of glutaradehyde (GA) and (3-aminopropyl) trimethoxysilane (APTES). Based on the number of specific recognition and binding between antibody (AbIGF-1) and antigen (AgIGF-1), the LC film exhibits gradual change from dark to bright under crossed polarizer. Under optimized conditions, the constructed LC-based biosensor demonstrates high sensitivity to IGF-I with a detection limit (LOD) of 1.0 ng/mL and a linear range of 10.0–2000.0 ng/mL (R2 = 0.989). This detection method possesses the advantages of easy construction, rapid sensing, good stability and repeatability, and high specificity for distinguishing IGF-I from other interfering proteins. Meanwhile, the format of biosensor could quickly detect IGF-I within a physiological range, providing profound information for prevention and treatment of GH-IGF-I axis related diseases in clinical diagnosis.

High-sensitive photometric microplate assay for tumor necrosis factor-alpha based on Fe@BC nanozyme

Optical immunoassay for the detection of protein biomarker tumor necrosis factor-α (TNF-α) is reported based on core–shell Fe@BC structure nanozyme as label. The nanozyme is composed of Fe nanoparticles as core and carbon layer as shell with element B doped into the carbon shell. The nanozyme displays good peroxidase-like activity that can oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to oxTMB in the presence of H2O2, generating blue colored compound. The detection of TNF-α was following the ELISA protocol by replacing traditional enzyme horseradish peroxidase (HRP) with nanozyme Fe@BC. The assay can be finished by about 2 h and has wide linear range for TNF-α from 5 pg/mL to 1 ng/mL with limit of detection of 2 pg/mL. The assay was applied for the detection of TNF-α in human serum samples with detection results comparable to commercial ELISA.

A review of biosensors for the detection of B-type natriuretic peptide as an important cardiovascular biomarker.

Heart disease, as the most serious threat to human health globally, is responsible for rising mortality rates, largely due to lifestyle and diet. Unfortunately, the main problem for patients at high risk of heart disease is the validation of prognostic tests. To this end, the detection of cardiovascular biomarkers has been employed to obtain pathological and physiological information in order to improve prognosis and early-stage diagnosis of chronic heart failure. Short-term changes in B-type natriuretic peptide are known as a standard and important biomarker for diagnosis of heart failure. The most important problem for detection is low concentration and short half-life in the blood. The normal concentration of BNP in blood is less than 7 nM (25 pg/mL), which increases significantly to more than 80 pg/mL. Therefore, the development of new biosensors with better sensitivity, detection limit, and dynamic range than current commercial kits is urgently needed. This review classifies the biosensors designed for detection of BNP into electrochemical, optical, microfluidic, and lateral-flow immunoassay techniques. The review clearly demonstrates that a variety of immunoassay, aptasensor, enzymatic and catalytic nanomaterials, and fluorophores have been successfully employed for detection of BNP at low attomolar ranges. Dtection of B-type natriuretic peptide with biosensors.

The determination of intact β-casein in milk products by biosensor immunoassay

A label-free optical biosensor immunoassay exploiting surface plasmon resonance detection for the estimation of the β-casein content in bovine milk and milk products is described. Samples were prepared by direct dilution with buffer and the protein was detected, under direct assay conditions, through binding with a commercially available anti-β-casein polyclonal antibody immobilised on the sensor surface. Assay conditions, selectivity and the potential for non-specific binding were defined. Analytical performance parameters included a method detection limit of 2.3 mg mL−1 for fluid milk, an intermediate precision RSDiR of 10.7 % for a skim milk powder and a mean recovery of 101.7 %, with a single functionalised flow cell being stable for at least 200 cycles. The method was found to be rapid, sensitive, precise and accurate, and is reliable for a range of milk products containing intact β-CN, and provides a routine complement to alternative conventional immunoassay and separation-based methods.

"Add on" Dual-Modal Optical Immunoassay by Plasmonic Metal NP-Semiconductor Composites.

Staphylococcus aureus enterotoxins (SEs, involving SEA, SEB, SEC, SED, and SEE) are considered to be the common toxins causing food poisoning and are not allowed to be detected in food. Accurate and anti-interfering SE detection in a complex food matrix is urgently required for food safety. Dual-modal optical sensors are able to avoid mutual interference of optical signals and possess the advantages of high accuracy and sensitivity. Herein, Au nanobipyramids (Au NBPs) and persistent luminescence ZnGeGaO:Cr,Er,Yb nanoparticle (ZGGO NP) nanocomposites are fabricated using the SEC antibody/antigen as templates, which display enhanced persistent luminescence (PL) and surface-enhanced Raman scattering (SERS) strength. The enhanced PL of Au NBP-ZGGO NP nanocomposites is ascribed to plasmon-enhanced radiative transitions. It is first found that ZGGO NPs display unique upconversion fluorescence, which can be absorbed by Au NBPs and that they largely excite the intensive electromagnetic field for SERS enhancement. Dual-model optical immunoassay achieved anti-interfering and specific SEC detection with a limit of detection of 7.5 pg/mL for the PL signal and 8.9 pg/mL for the SERS signal in the range of 10 pg/mL-100 ng/mL. Depending on the plasmon-enhanced PL mechanism and upconversion fluorescence-enhanced SERS principle, plasmonic NP-semiconductor composites show potential prospects in the establishment of multimodal optical biosensors for the quantitative and accurate evaluation of analytes in a complex food matrix.

Duplex Shiny app quantification of the sepsis biomarkers C-reactive protein and interleukin-6 in a fast quantum dot labeled lateral flow assay

Fast point-of-care (POC) diagnostics represent an unmet medical need and include applications such as lateral flow assays (LFAs) for the diagnosis of sepsis and consequences of cytokine storms and for the treatment of COVID-19 and other systemic, inflammatory events not caused by infection. Because of the complex pathophysiology of sepsis, multiple biomarkers must be analyzed to compensate for the low sensitivity and specificity of single biomarker targets. Conventional LFAs, such as gold nanoparticle dyed assays, are limited to approximately five targets—the maximum number of test lines on an assay. To increase the information obtainable from each test line, we combined green and red emitting quantum dots (QDs) as labels for C-reactive protein (CRP) and interleukin-6 (IL-6) antibodies in an optical duplex immunoassay. CdSe-QDs with sharp and tunable emission bands were used to simultaneously quantify CRP and IL-6 in a single test line, by using a single UV-light source and two suitable emission filters for readout through a widely available BioImager device. For image and data processing, a customized software tool, the MultiFlow-Shiny app was used to accelerate and simplify the readout process. The app software provides advanced tools for image processing, including assisted extraction of line intensities, advanced background correction and an easy workflow for creation and handling of experimental data in quantitative LFAs. The results generated with our MultiFlow-Shiny app were superior to those generated with the popular software ImageJ and resulted in lower detection limits. Our assay is applicable for detecting clinically relevant ranges of both target proteins and therefore may serve as a powerful tool for POC diagnosis of inflammation and infectious events.

Rapid point of care test for detecting urogenital Chlamydia trachomatis infection in nonpregnant women and men at reproductive age.

Chlamydia trachomatis (C trachomatis) is one of the most frequent sexually transmitted infections and a source of deleterious effects on the reproductive health of men and women. Because this infection is likely asymptomatic and is associated with subfertility, ectopic pregnancy, and chronic pain, its presence needs to be confirmed. Technologies available for the diagnosis of C trachomatis infection can be classified into tests performed in a laboratory and rapid tests at the point of care (POC tests). Laboratory-based tests include culture, nucleic acid amplification tests, enzyme immunoassays (EIA), direct fluorescent antibody, nucleic acid hybridization, and transformation tests. Rapid tests include solid-phase EIA and solid-phase optical immunoassay. POC tests can be performed within 30 minutes without the need for expensive or sophisticated equipment. The principal advantage of this technology is the immediate presentation of results with the subsequent possibility to start the treatment of infected patients immediately. To determine the diagnostic accuracy of rapid point-of-care (POC) testing for detecting urogenital C trachomatis infection in nonpregnant women and men of reproductive age, as verified with nucleic acid amplification tests (NAATs) as the reference standard. In November 2019 we searched CENTRAL, MEDLINE, Embase and LILACS. We also searched Web of Science, two trials registries and an abstract database. We screened reference lists of included studies for additional references. We included diagnostic accuracy studies of symptomatic or asymptomatic nonpregnant women and men reproductive age. Included trials should have prospectively enrolled participants without previous diagnostic testing, co-infections or complications and consecutively or through random sampling at primary or secondary care facilities. Only studies reporting that all participants received the index test and the reference standard and presenting 2 x 2 data were eligible for inclusion. We excluded diagnostic case-control studies. Two review authors independently screened titles and abstracts for relevance. Two review authors independently, and in duplicate, assessed eligibility, extracted data, and carried out quality assessment. We resolved differences through consensus or by involving a third review author. We assessed studies for methodological quality using QUADAS-2 and used meta-analysis to combine the results of studies using the bivariate approach to estimate the expected sensitivity and specificity values. We assessed the quality of the evidence using GRADE criteria and explored sources of heterogeneity. We included a total of 19 studies, with 13,676 participants, that assessed the diagnostic accuracy of POC tests for C trachomatis infection in nonpregnant women and men of reproductive age, as verified with NAATs as the reference standard. Rapid tests were provided by the distributors in nine studies. Seven studies recruited a predominantly high risk or symptomatic population; the studies were conducted in America, Asia, Africa, Europe and Oceania, with a median prevalence of 10% (range 8% to 28%); nine different brands were assessed. The mean sensitivity for rapid tests for detecting urogenital infection was 0.48 (95% confidence interval (CI) 0.39 to 0.58; low-quality evidence) with a mean specificity of 0.98 (95% CI 0.97 to 0.99; moderate-quality evidence). We explored sources of heterogeneity by looking into differences in diagnostic accuracy according to the specimen (endocervical versus urine or vaginal), symptoms among participants (symptomatic versus asymptomatic), and setting (low/middle-income versus high-income countries). Likelihood ratio tests were not significantly different in terms of sensitivity or specificity by specimen (P = 0.27) or setting (P = 0.28); for this reason, these covariates do not appear to explain the observed variability. Included studies did not provide enough information to assess the 'presence of symptoms' covariate. We downgraded the quality of evidence because of some limitations in applicability and heterogeneity. Based on the results of this systematic review, the POC test based on antigen detection has suboptimal sensitivity but good specificity. Performance of this test translates, on average, to a 52% chance of mistakenly indicating absence of infection and a 2% chance of mistakenly pointing to the presence of this condition. Because of its deleterious consequences for reproductive health, and considering the current availability of safe and effective interventions to treat C trachomatis infection, the POC screening strategy should not be based on a rapid diagnostic test for antigen detection. Research in this topic should focus on different technologies.

A simple and fast protocol for the synthesis of 2-amino-4-(4-formylphenyl)-4H-chromene-3-carbonitrile to develop an optical immunoassay for the quantification of botulinum neurotoxin type F

In this study, a novel optical immunoassay platform using (S)-2-amino-4-(4-formylphenyl)-4H-chromene-3-carbonitrile, which was synthesized by an ultra-sonication method, as an optical probe.

Magnetic characterization change by solvents of magnetic nanoparticles in liquid-phase magnetic immunoassay

Liquid-phase magnetic immunoassay (MIA) using magnetic nano-particles (MNPs) has been studied as a more rapid method compared to optical methods for inspecting proteins and viruses. MIA can estimate the number of conjugated antibodies without being washed differently from conventional optical immunoassay. However, in the case of the liquid phase, it is considered that the magnetic properties of MNPs are affected by physical properties such as viscosity and impurity substances such as biological substances contained in the blood. In this study, the effect of sodium chloride (NaCl) in buffer and serum solution was evaluated to reveal the effect of serum because the sodium (Na+) and chloride (Cl-) ions in the serum dominate ion balance of blood. The measurement results of AC magnetic susceptibility and a dynamic light scattering (DLS) showed that the aggregation of MNPs was largely affected by the concentration of NaCl. This effect of the NaCl could be explained by shielding of the surface charge of MNPs by ions in the solution. Although the concentrations of NaCl in the buffer and serum solution were almost same, we found that MNPs were aggregated more in their size for those in the serum solution because of other impurities, such as proteins. These results suggest evaluation of effects of the contaminants in serum and optimization of polymer coatings of MNPs could be important factors to realize measurements of magnetic immunoassay with high accuracy.