The clinical value of high mobility group box-1 and CRP/Alb ratio in the diagnosis and evaluation of sepsis in children.

OBJECTIVE: To explore the clinical value of high mobility group box-1 (HMGB-1), C-reactive protein (CRP), procalcitonin (PCT), and CRP to albumin (Alb) ratio in the diagnosis and evaluation of the severity of sepsis in children. PATIENTS AND METHODS: A total of 90 children, 50 with sepsis and 40 with general infection, whose symptoms did not meet the criteria for diagnosis of sepsis, were admitted to the Pediatrics Department of Jingzhou Central Hospital in Hubei Province between November 2021 and December 2022, were enrolled and selected as experimental and control group, respectively. The serum of two groups was collected within 24 hours after admission, the levels of HMGB-1 were detected by enzyme-linked immunosorbent assay (ELISA), and CRP, PCT, Alb, and hospitalization days were recorded. The differences in indicators between the two groups were compared, and correlation analysis was performed between hospitalization days and various indicators. The receiver operating characteristic (ROC) curve was drawn to evaluate the independent or combined value of CRP, PCT, HMGB-1, and CRP/Alb ratio in the early diagnosis of sepsis in children. RESULTS: These four indicators of children with sepsis were significantly higher than those in the general infection group (all p=0.000). The levels of CRP, PCT and CRP/Alb ratio were significantly positively correlated with the hospitalization days (r=0.329, 0.333, 0.329; p=0.02, 0.01, 0.002). The area under curve (AUC) of CRP, PCT, HMGB-1, and CRP/Alb ratio for the diagnosis of sepsis in children was 0.798, 0.817, 0.838, 0.809, respectively, and that of the combination of four indicators was 0.952. CONCLUSIONS: CRP, PCT, HMGB-1, and CRP/Alb ratio resulted as effective indicators for early diagnosis and evaluation of childhood sepsis, having a higher value in combined diagnosis.

Rapid FRET-based homogeneous immunoassay of procalcitonin using matched carbon dots labels.

A novel method for the detection of procalcitonin in a homogeneous system by matched carbon dots (CDs) labeled immunoprobes was proposed based on the principle of FRET and double antibody sandwich method. Blue-emitting carbon dots with a strong fluorescence emission range of 400-550 nm and red-emitting carbon dots with the best excitation range of 410-550 nm were prepared before they reacted with procalcitonin protoclone antibody pairs to form immunoprobes. According to the principles of FRET, blue-emitting carbon dots were selected as the energy donor and red-emitting carbon dots as the energy receptor. The external light source excitation (310 nm) could only cause weak luminescence of CDs. However, once procalcitonin was added, procalcitonin and antibodies would be combined with each other quickly (≤20 min). Here, blue-emitting carbon dots acquired energy could be transferred to red-emitting carbon dots efficiently, causing the emitted fluorescence enhancement of red-emitting carbon dots. The fluorescence detection results in PBS buffer solution and diluted rabbit blood serum showed that the fluorescence intensity variation was linear with the concentration of procalcitonin. There was a good linear relationship betweenF/F0 and procalcitonin concentrations in PBS buffer solution that ranged from 0 to 100 ng ml-1, and the linear equation wasF/F0 = 0.004 *Cpct + 0.98359. Detection in the diluted rabbit serum led to the results that were linear in two concentration ranges, including 0-40 ng ml-1and 40-100 ng ml-1, and the detection limit based on 3σK-1was 0.52 ng ml-1. It is likely that this matched CDs labeled immunoprobes system can provide a new mode for rapid homogeneous detection of disease markers.

A dual signal-amplified electrochemiluminescence immunosensor based on core-shell CeO2-Au@Pt nanosphere for procalcitonin detection.

A dual signal-amplified sandwich electrochemiluminescence (ECL) immunosensor was fabricated for trace detection of procalcitonin (PCT). CeO2-Au@Pt composed of sea urchin-like Au@Pt nanoparticles coated on CeO2 hollow nanospheres was immobilized on electrode surface to electrochemically catalyze H2O2 to produce a large number of superoxide anion (O2•-). The immunosensor was prepared by linking the capture antibody on immobilized CeO2-Au@Pt with heptapeptide (HWRGWVC), which could maintain the activity of the antibody. The prepared Au star@BSA was used to bind abundant luminol for labeling the secondary antibody (Ab2). Upon the sandwich-typed immunoreactions, the O2•- could react with the introduced luminol on the immunosensor surface to produce strong ECL intensity. With an outstanding linear detection range and a low detection limit of 17 fg/mL, the ECL immunosensor permitted ultrasensitive detection of PCT at a low H2O2 concentration and demonstrated its high application potential in the clinical assay.

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration.

The quantification of low abundant proteins in complex matrices by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains challenging. A measurement procedure based on optimized antibody-free sample preparation and isotope dilution coupled to LC-MS/MS was developed to quantify procalcitonin (PCT) in human serum at sub-microgram per liter level. A combination of sodium deoxycholate-assisted protein precipitation with acetonitrile, solid-phase extraction, and trypsin digestion assisted with Tween-20 enhanced the method sensitivity. Linearity was established through peptide-based calibration curves in the serum matrix (0.092-5.222 µg/L of PCT) with a good linear fit (R2 ≥ 0.999). Quality control materials spiked with known amounts of protein-based standards were used to evaluate the method's accuracy. The bias ranged from -2.6 to +4.3%, and the intra-day and inter-day coefficients of variations (CVs) were below 2.2% for peptide-based quality controls. A well-characterized correction factor was determined and applied to compensate for digestion incompleteness and material loss before the internal standards spike. Results with metrological traceability to the SI units were established using standard peptide of well-characterized purity determined by peptide impurity corrected amino acid analysis. The validated method enables accurate quantification of PCT in human serum at a limit of quantification down to 0.245 µg/L (bias -1.9%, precision 9.1%). The method was successfully applied to serum samples obtained from patients with sepsis. Interestingly, the PCT concentration reported implementing the isotope dilution LC-MS/MS method was twofold lower than the concentration provided by an immunoassay.

Procalcitonin measurement using antibody-conjugated fluorescent microspheres distinguishes atypical bacterial meningitis from viral encephalitis in children.

Examination of cerebrospinal fluid in atypical bacterial meningitis (ABM) is similar to that of viral encephalitis (VE), so ABM can easily be misdiagnosed as VE, which can delay diagnosis and treatment. We developed a simple, rapid hand-held lateral flow immunoassay detection system based on fluorescent microspheres (FMS) for procalcitonin (PCT) detection, which provides an indicator to differentiate between ABM and VE. With this novel method, the antigen-antibody reaction systems involve different species, making the test strips more stable than those utilizing one species. The strips exhibited a wide dynamic range (0.04-50 ng/mL) and good sensitivity (0.03 ng/mL). The function of PCT in the identification of ABM and VE in children was further studied. A significant difference in PCT levels was observed between the ABM and VE groups (P = 0.00) and between the ABM and the normal control groups (P = 0.00). PCT levels were not significantly different between the VE and normal control groups (P = 0.30). The area under the receiver operating characteristic curve of PCT for the diagnosis of ABM was 0.95. These findings collectively indicate the usefulness of the PCT detection method based on FMS for clinically differentiating between ABM and VE.

Fiber optic nanogold-linked immunosorbent assay for rapid detection of procalcitonin at femtomolar concentration level.

A rapid and ultrasensitive biosensing method based on fiber optic nanogold-linked immunosorbent assay is reported. The method employs an immobilized capture probe on the fiber core surface of an optical fiber and a detection probe conjugated to gold nanoparticles (AuNPs) in a solution. Introduction of a sample containing an analyte and the detection probe into a biosensor chip leads to the formation of a sandwich-like complex of capture probe-analyte-detection probe on the fiber core surface, through which nanoplasmonic absorption of the fiber optic evanescent wave occurs. The performance of this method has been evaluated by its application to the detection of procalcitonin (PCT), an important biomarker for sepsis. In this study, anti-PCT capture antibody is functionalized on an unclad segment of an optical fiber to yield a fiber sensor and anti-PCT detection antibody is conjugated to AuNPs to afford nanoplasmonic probes. The method provides a wide linear response range from 1 pg/mL to 100 ng/mL (5 orders) and an extremely low limit of detection of 95 fg/mL (7.3 fM) for PCT. In addition, the method shows a good correlation in determining PCT in blood plasma with the clinically validated electrochemiluninescent immunoassay. Furthermore, the method is quick (analysis time ≤15 min), requires low-cost instrumentation and sensor chips, and is also potentially applicable to the detection of many other biomarkers.

Electrochemiluminescence Double Quenching System Based on Novel Emitter GdPO4:Eu with Low-Excited Positive Potential for Ultrasensitive Procalcitonin Detection.

Nowadays, the electrochemiluminescence (ECL) immunosensor with the unique superiority of tunable luminescence and ultrahigh sensitivity has become one of the most promising immunoassay techniques, especially for low-abundance biomarkers analysis. However, the use of signal probes with high excited potential and applied emitters which owned good intensity but biotoxicity limited its application. Herein, an ECL resonance energy transfer strategy was developed based on protein bioactivity protection utilizing europium-doped phosphoric acid gadolinium (GdPO4:Eu) as novel low-potential luminophor (donor) and Pd@Cu2O as the quenching probe (acceptor). Specifically, GdPO4:Eu was first prepared by using the hydrothermal synthesis method to apply in ECL, and when it coexisted with K2S2O8, cathode, a strong ECL signal would be generated at a low potential of -1.15 V (vs Ag/AgCl), where the immunocompetence of antigens and antibodies can be maintained well. Electrical pair Eu3+/Eu2+, as the coreactant promoter, produced by potential excitation could produce more SO4•- to accelerate the oxidation process of GdPO4:Eu. Meanwhile, Cu2O coated onto Pd (Pd@Cu2O), as a dual-quencher, enhanced the quenching effect of Pd alone and controlled the ECL intensity of the "signal on" state within a reasonable range. As a result, the proposed biosensor for detection of trace procalcitonin, a biomarker of systemic inflammatory response syndrome, exhibited a far low detection limit, 0.402 fg/mL (S/N = 3). Importantly, this work not only utilized a promising ECL emitter for biosensing platform construction but also had momentous potential in biomarker detection of disease diagnosis and clinical analysis.

Highly-branched Cu2O as well-ordered co-reaction accelerator for amplifying electrochemiluminescence response of gold nanoclusters and procalcitonin analysis based on protein bioactivity maintenance.

The point of fabricating ultrasensitive electrochemiluminescence (ECL)-based biosensors should be focused on how to maintain high immune recognition of antigens by antibodies in whole process. That is not effortless due to the structure of the protein can be destroyed root in toxic nanocarriers, excessive cyclic potential and superoxide radicals in coreactant, all of which can lead to reduce the bioactivity of antigen and antibody. In this work, the effect of negative voltage and divers coreactant on protein bioactivity were verified. Based on that, a motivated ECL biosensor with good biocompatibility was fabricated for procalcitonin (PCT) detection using Au nanoclusters (Au NCs) as low-potential cathodic luminophor and K2S2O8 as non-toxic coreactant, respectively. Besides, highly-branched Cu2O was utilized to catalyze K2S2O8 and produce more radical anion SO4•-, which can oxidize Au NCs•- to generate more high-energy-state Au NCs*, thus doubling the ECL intensity to meet the requirements of trace analysis. In addition, protein A (PA) as specific antibody capturer was employed to bind the Fc region of anti-PCT in an orientated way, further maintaining the physiological activity of antibody. As expected, all strategies undoubtedly practically improved the immune recognition of the biosensor and reduced the detection limit to 2.90 fg/mL.

Time-Resolved Digital Immunoassay for Rapid and Sensitive Quantitation of Procalcitonin with Plasmonic Imaging.

Timely diagnosis of acute diseases improves treatment outcomes and saves lives, but it requires fast and precision quantification of biomarkers. Here, we report a time-resolved digital immunoassay based on plasmonic imaging of binding of single nanoparticles to biomarkers captured on a sensor surface. The real-time and high contrast of plasmonic imaging lead to fast and precise counting of the individual biomarkers over a wide dynamic range. We demonstrated the detection principle, evaluated the performance of the method using procalcitonin (PCT) as an example, and achieved a limit of detection of ∼2.8 pg/mL, dynamic range of 4.2-12500 pg/mL, for a total detection time of ∼25 min.

An ultrasensitive electrochemical immunosensor for procalcitonin detection based on the gold nanoparticles-enhanced tyramide signal amplification strategy.

In this study, we established an ultrasensitive electrochemical immunosensor based on the gold nanoparticles-enhanced tyramide signal amplification (AuNPs-TSA) for the detection of procalcitonin (PCT, for discriminating bacterial infections from nonbacterial infections). Firstly, a facilely prepared, well-conducting reduced graphene oxide nanosheets/GNP (rGO-AuNPs) nanocomposite was synthesized and immobilized on the electrode surface to absorb more capture antibodies (Ab1). Next another nanocomposite, acting as a signal tool, was modified with detection antibody (Ab2) and horseradish peroxidase (HRP), and then backfilled by bovine serum albumin (BSA). Because a single AuNP is able to load multiple HRPs and BSAs, a number of tyramine labeled biotins (T-B) could be deposited on the proteins adhering to the surface of AuNPs. Moreover, the high affinity between streptavidin (SA) and biotins significantly increases the loading of streptavidin labeled horseradish peroxidase (SA-HRP). The amplification system which was based on the two nanocomposites mentioned above, effectively amplified the electric current signals. This immunosensor exhibits a wide dynamic detection range from 0.05 ng mL-1 to 100 ng mL-1 and with an ultralow detection limit of 0.1 pg mL-1. We have successfully utilized this immunosensor to quantify the concentration of PCT in human serum samples, and the results suggest its potential use in clinical application.

Label-Free Sandwich Imaging Ellipsometry Immunosensor for Serological Detection of Procalcitonin.

Analysis of trace low molecular weight (LMW) proteins in serum using the label-free imaging ellipsometry (IE) immunosensor is still a challenge due to the lack of an effective signal amplification strategy and the serious nonspecific adsorption. Herein we have developed a sandwich strategy-mediated IE (SSIE) immunosensor to enable the immunodetection of LMW protein biomarkers in serum samples. We have first found that the weak binding affinity and the insufficient surface amount density of the ligand are two important factors which hinder the detection of LMW proteins in serum using the IE immunosensor. Then we have deduced that the sandwich strategy can amplify the detection signal of IE and avoid the nonspecific adsorption in serum. As a validation of the serological detection of LMW proteins, the SSIE immunosensor has been used to accomplish the quantitative detection of procalcitonin (PCT) in serum. Compared with other PCT analysis methodologies, the SSIE immunosensor enjoys the advantages of simplicity, rapidity, and sufficient sensitivity. Furthermore, we have proposed the criteria to predict the ability of the SSIE immunosensor for the detection of LMW protein biomarkers in serum, which can make the detection of LMW proteins smart and efficient.

Comparison of the Temperature Influence on the Activity of Currently Available Procalcitonin Reagents.

BACKGROUND: Procalcitonin (PCT) is a stable biomarker for bacterial infections; however, limited data is available on new trivalent reagents. We evaluated temperature influence on the activity of PCT reagents. METHODS: Using both conventional and trivalent reagents, we measured PCT levels of 30 clinical samples, stored residuum at refrigerator (4°C) and room temperature (24°C), and reexamined it after 24 hours. We defined a reduction rate as a percentage of PCT level at 24 hours compared to that after defrost and evaluated a ratio of reduction rate in 4°C to that in 24°C. RESULTS: The reduction rate at room temperature decreased significantly compared to that in the refrigerated condition for all the reagents examined (p < 0.001). In addition, the ratio of reduction rate between the conventional and trivalent reagents showed a significant difference (p < 0.001). CONCLUSIONS: The serum PCT levels significantly decrease at room temperature, particularly when using newer trivalent reagents.