Biomarkers In Human Serum Research Articles

Highly Sensitive Multiplexed Sensing of miRNAs in a Gastric Cancer Patient's Liquid Biopsy.

Gastric cancer (GC) is one of the leading causes of cancer mortality in the world. Most patients are in the advanced stage of the disease at the time of diagnosis because the symptoms of early gastric cancer patients are not obvious. Early diagnosis of gastric cancer is still challenging due to the high cost, invasiveness, and low accuracy of traditional diagnostic methods such as endoscopy and biopsy. Herein, we develop clinically accurate and highly sensitive detection of multiple GC miRNA biomarkers in human serum using an isothermal nucleic acid primer exchange reaction (PER). The isothermal nucleic acid primer exchange reaction demonstrates high sensitivity and robustness, exemplified by a one-pot reaction achieving a detection limit of 28.71 fM. By quantifying the levels of three miRNA biomarkers selected through bioinformatics analysis in gastric cancer serum samples, the diagnostic approach effectively distinguished between clinical gastric cancer patients (n = 25) and noncancer controls (n = 10). The performance of our three-miRNA signature in discriminating between GC and controls was as follows: area under the curve (AUC): 0.808, sensitivity: 89%, specificity: 88%, positive predictive value (PPV): 96%, and negative predictive value (NPV): 70%.

Laser-Induced Electrochemical Biosensor Modified with Graphene-Based Ink for Label-Free Detection of Alpha-Fetoprotein and 17β-Estradiol.

In this research, a novel electrochemical biosensor is proposed based on inducing graphene formation on polyimide substrate via laser engraving. Graphene polyaniline (G-PANI) conductive ink was synthesized by planetary mixing and applied to the working zone of the developed sensor to effectively enhance the electrical signals. The laser-induced graphene (LIG) sensor was used to detect alpha-fetoprotein (AFP) and 17β-Estradiol (E2) in the phosphate buffer saline (PBS) buffer and human serum. The electrochemical performance of the biosensor in determining these biomarkers was investigated by differential pulse voltammetry (DPV) and chronoamperometry (CA). In a buffer environment, alpha-fetoprotein (AFP) and 17β-Estradiol detection range were 4-400 ng/mL and 20-400 pg/mL respectively. The experimental results showed a limit of detection (LOD) of 1.15 ng/mL and 0.96 pg/mL for AFP and estrogen, respectively, with an excellent linear range (R2 = 0.98 and 0.99). In addition, the designed sensor was able to detect these two types of biomarkers in human serum successfully. The proposed sensor exhibited excellent reproducibility, repeatability, and good stability (relative standard deviation, RSD = 0.96%, 1.12%, 2.92%, respectively). The electrochemical biosensor proposed herein is easy to prepare and can be successfully used for low-cost, rapid detection of AFP and E2. This approach provides a promising platform for clinical detection and is advantageous to healthcare applications.

Integrated biosensor array for multiplex biomarkers cancer diagnosis via in-situ self-assembly carbon nanotubes with an ordered inverse-opal structure

To enhance the precision and reliability of early disease detection, especially in malignancies, an exhaustive investigation of multi-target biomarkers is essential. In this study, an advanced integrated electrochemical biosensor array that demonstrates exceptional performance was constructed. This biosensor was developed through a controllable porous-size mechanism and in-situ modification of carbon nanotubes (CNTs) to quantify multiplex biomarkers—specifically, C-reaction protein (CRP), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA)—in human serum plasma. The fabrication process involved creating a highly ordered three-dimensional inverse-opal structure with the CNTs (pCNTs) modifier through microdroplet-based microfluidics, confined spatial self-assembly of nanoparticles, and chemical wet-etching. This innovative approach allowed for direct in-situ modification of nanomaterial onto the surface of electrode array, eliminating secondary transfer and providing exceptional control over structure and stability. The outstanding electrochemical performance was achieved through the synergistic effect of the pCNTs nanomaterial, aptamer, and horseradish peroxidase-labeled (HRP-) antibody. Additionally, the integrated biosensor array platform comprised multiple individually addressable electrode units (n = 11), enabling simultaneous multi-parallel/target testing, thereby ensuring accuracy and high throughput. Crucially, this integrated biosensor array accurately quantified multiplex biomarkers in human serum, yielding results comparable to commercial methods. This integrated technology holds promise for point-of-care testing (POCT) in early disease diagnosis and biological analysis.

Enhancing biomarker detection in human serum for lung cancer diagnosis: Aqueous biphasic systems for simultaneous depletion of high-abundance proteins and efficient extraction of CYFRA 21–1

Analysing biomarkers in human serum could be used as an effective and less invasive approach for the diagnosis of lung cancer; however, biomarker detection reliability is highly limited due to matrix effects. Herein, aqueous biphasic systems (ABS) are studied as platforms for human serum pretreatment, allowing the simultaneous depletion of high abundant proteins and biomarker extraction. By using ABS varying the polyethylene glycol (PEG) molecular weight between 400 and 6000 g·mol−1 and adopting phosphate buffer as the other phase-forming component, the depletion of the high abundance serum proteins immunoglobulin G (IgG) and human serum albumin (HSA) is induced at the ABS interphase, through precipitation, forming a three-phase partitioning system (ABS-TPP). Maximum depletion efficiencies of 99 % for IgG and 70 % for HSA were achieved in one step using PEG 1500-based ABS-TPP. On the other hand, lung cancer biomarkers, such as CYFRA 21–1, are extracted to the PEG-rich phase of the same ABS-TPP with recovery yields of 91 %. This work shows that a proper selection of the PEG molecular weight in the ABS composition leads to the efficient depletion of high-abundance proteins and extraction of cancer biomarkers from human serum, in a single step, confirming the potential of ABS for sample pretreatment to improve biomarker analysis.

A Lateral-Flow Device for the Rapid Detection of Scedosporium Species.

Scedosporium species are human pathogenic fungi, responsible for chronic, localised, and life-threatening disseminated infections in both immunocompetent and immunocompromised individuals. The diagnosis of Scedosporium infections currently relies on non-specific CT, lengthy and insensitive culture from invasive biopsy, and the time-consuming histopathology of tissue samples. At present, there are no rapid antigen tests that detect Scedosporium-specific biomarkers. Here, we report the development of a rapid (30 min) and sensitive (pmol/L sensitivity) lateral-flow device (LFD) test, incorporating a Scedosporium-specific IgG1 monoclonal antibody (mAb), HG12, which binds to extracellular polysaccharide (EPS) antigens between ~15 kDa and 250 kDa secreted during the hyphal growth of the pathogens. The test is compatible with human serum and allows for the detection of the Scedosporium species most frequently reported as agents of human disease (Scedosporium apiospermum, Scedosporium aurantiacum, and Scedosporium boydii), with limits of detection (LODs) of the EPS biomarkers in human serum of ~0.81 ng/mL (S. apiospermum), ~0.94 ng/mL (S. aurantiacum), and ~1.95 ng/mL (S. boydii). The Scedosporium-specific LFD (ScedLFD) test therefore provides a potential novel opportunity for the detection of infections caused by different Scedosporium species.

Low fouling electrochemical sensing interfaces based on Bovine serum albumin hydrogel for the detection of protein biomarkers in human serum

The development of electrochemical biosensor capable of detecting specific biomarkers in real complex biological samples remains a challenge significantly relevant to a range of disease diagnostic applications. Herein an antifouling electrochemical biosensor suitable for biomarker detection in human serum was constructed based on protein hydrogels. A typical protein bovine serum albumin (BSA) was crosslinked with glutaraldehyde to form a hydrogel, which was freeze-dried and re-swollen to exhibit excellent antifouling capability. The rheological testing revealed that the prepared BSA hydrogel possessed good elasticity with a critical strain value of about 600 %, which is important for the construction of stable hydrogel-based biosensors. Following the immobilization of CA125 antibodies onto the BSA hydrogel modified electrode, an electrochemical biosensor for the detection of CA125 was constructed, with a linear range of 0.1 ∼ 1000 U mL−1 and a limit of detection of 0.026 U mL−1. Owing to the unique properties of the BSA hydrogel, the biosensor exhibited good stability and excellent antifouling performances, and it was able to detect CA125 in human serum with acceptable accuracy. The strategy of constructing low fouling biosensors based on hydrogels prepared from BSA with biocompatibility and widespread availability represented a very promising advance along the road to develop biosensing devices with robust operation in complex biological fluids.

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Serum Autoantibody Biomarkers for Management of Rheumatoid Arthritis Disease.

Rheumatoid arthritis (RA) is a systemic chronic autoimmune inflammatory disease that is characterized by the destruction of bone and production of autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies (ACPAs). The high prevalence of this disease and the need of affordable tools for its early detection led us to prepare the first electrochemical immunoplatform for the simultaneous determination of four RA biomarkers, the autoantibodies: RF, anti-peptidyl-arginine deiminase enzyme (anti-PAD4), anti-cyclic citrullinated peptide (anti-CCP), and anti-citrullinated vimentin (anti-MCV). Functionalized magnetic beads (MBs) were used to immobilize the specific antigens, and sandwich-type immunoassays were implemented for the amperometric detection of the four autoantibodies, using the horseradish peroxidase (HRP)/H2O2/hydroquinone (HQ) system. The immunoplatform was applied to the determination of the biomarkers in human serum of twenty-two patients diagnosed with RA and four healthy individuals, and the results were validated against ELISA tests and the certified values.

3D-Printed Electrochemical Platform for Detection of Diabetes Biomarkers: Drop-Based and Time-Based Readout for Clinical Diagnosis

Multiplex detection of biomarkers in clinical matrices on a single sensor platform is the need for a detailed disease diagnosis and patient prognosis. Herein, we demonstrate immunoassay/enzyme detection assay formats on a 3D-printed device for measuring insulin, C peptide, and glucose in two-fold diluted human serum electrochemically. The sensor platform comprised of a graphene–gold (Gr–Au) framework to immobilize insulin and C peptide specific antibodies and glucose oxidase to detect clinically relevant levels of diabetes biomarkers with good sensitivity, specificity, and reproducibility. The fabrication of the 3D-printed sensor platform is characterized by field emission scanning electron microscopy (FESEM) and electron impedance spectroscopy (EIS). The current signals for detection of free serum insulin, C peptide were monitored by oxidation of ferro/ferricyanide redox probe, while glucose was detected directly using square wave voltammetry (drop-based) and amperometry (real-time). The designed electrochemical platform offered a limit of detection (LOD) 4 pM insulin, 0.02 nM C peptide, and 0.05 mM glucose. The Gr–Au framework was ~ 2.5× more sensitive in detecting diabetes biomarkers in human serum when compared to pristine Gr framework. Additionally, the sensor does not show any cross-reactivity to non-specific biomarkers in human serum.

A novel photoelectrochemical microfluidic chip for multi-index determination of diabetes and its complications

Rapid and accurate monitoring of glucose, lactic acid, pyruvic acid, and 3-hydroxybutyric acid is essential in preventing, diagnosing, and treating diabetes, lactic acidosis and diabetic ketoacidosis. Herein, a novel sensing chip for multi-index determination of diabetes, lactic acidosis, and diabetic ketoacidosis was presented by integrating microfluidic device and photoelectrochemical (PEC) sensor. In order to block the interference from the reductive species in real samples, the PEC sensor was divided into a biocathode and a photoanode, which were installed separately in the upper and bottom layers of the device. The photoanodes were modified with ZnIn2S4 nanoflower as photosensitive material, while enzymes for catalyzing the analytes were immobilized on the biocathodes. The PEC chip displayed wide detection ranges with low detection limits of 0.035 μM, 0.34 μM, 3.3 μM and 0.035 μM for the four analytes (S/N = 3). The chip also demonstrated decent anti-interference capability and reliability in monitoring the four biomarkers in human serum. Furthermore, a household amperemeter was deployed to record the photocurrent signals, which helps to reduce the cost. By replacing the enzyme on the biocathode, the sensing chip could play a role in monitoring a broad range of species.

Click reaction-assisted construction of antifouling immunosensors for electrochemical detection of cancer biomarkers in human serum

Herein, an electrochemical immunosensor capable of detecting targets in human serum with ultralow fouling and high sensitivity was successfully constructed through a click reaction. One designed biotinylated peptide was associated with the other peptide functionalized with biotin and azide groups at its two terminals, to form a branched antifouling peptide through the streptavidin-biotin affinity interaction, and it was attached to the electrode to construct an antifouling interface. Then, the exposed azide group on the branched peptide was used to link the antibody modified with 5′-dibenzocyclooctyne (DBCO), and the immunosensor was thus constructed with the aid of the click reaction between the azide group and the DBCO. The developed antifouling immunosensor demonstrated a wide linear response range for carcinoembryonic antigen (CEA) and a low limit of detection of 40 fg mL−1, which is much superior to the sensing performance of the immunosensor prepared through the normal EDC/NHS method. The immunosensor was capable of assaying CEA in undiluted serum samples with satisfying accuracy when compared with a clinically approved method. This work offered an effective strategy to develop various biosensors through the conjugation of antifouling peptides with different antibodies.

Long-term stability and age-dependence of six regulatory serum proteins.

Aim: The development of biomarker-based diagnostic procedures often relies on samples stored for several years. We aimed to investigate the influence of storage time and patient age on six neuroregulatory and immunoregulatory serum biomarkers. Materials & methods: We quantified six biomarkers in serum from 151 individuals using ELISA. Serum was stored at -80°C for up to 9.5years. Results: When associating storage time with biomarker values, BDNF, VEGF-A and TGF-β1 showed a significant increase over time; IGF-1, MCP-1 and IL-18 did not. Associating participant age with biomarkers, only IL-18 in Alzheimer's disease patients showed a significant increase. Conclusion: Storage time can influence results of biomarkers in human serum. This needs to be considered when assessing samples stored for several years.

SERS Based Lateral Flow Assay for Rapid and Ultrasensitive Quantification of Dual Laryngeal Squamous Cell Carcinoma-Related miRNA Biomarkers in Human Serum Using Pd-Au Core-Shell Nanorods and Catalytic Hairpin Assembly.

Non-invasive early diagnosis is of great significant in disease pathologic development and subsequent medical treatments, and microRNA (miRNA) detection has attracted critical attention in early cancer screening and diagnosis. However, it was still a challenge to report an accurate and sensitive method for the detection of miRNA during cancer development, especially in the presence of its analogs that produce intense background noise. Herein, we developed a surface-enhanced Raman scattering (SERS)–based lateral flow assay (LFA) biosensor, assisted with catalytic hairpin assembly (CHA) amplification strategy, for the dynamic monitoring of miR-106b and miR-196b, associated with laryngeal squamous cell carcinoma (LSCC). In the presence of target miRNAs, two hairpin DNAs could self-assemble into double-stranded DNA, exposing the biotin molecules modified on the surface of palladium (Pd)–gold (Au) core–shell nanorods (Pd-AuNRs). Then, the biotin molecules could be captured by the streptavidin (SA), which was fixed on the test lines (T1 line and T2 line) beforehand. The core–shell spatial structures and aggregation Pd-AuNRs generated abundant active “hot spots” on the T line, significantly amplifying the SERS signals. Using this strategy, the limits of detections were low to aM level, and the selectivity, reproducibility, and uniformity of the proposed SERS-LFA biosensor were satisfactory. Finally, this rapid analysis strategy was successfully applied to quantitatively detect the target miRNAs in clinical serum obtained from healthy subjects and patients with LSCC at different stages. The results were consistent with the quantitative real-time PCR (qRT-PCR). Thus, the CHA-assisted SERS-LFA biosensor would become a promising alternative tool for miRNAs detection, which showed a tremendous clinical application prospect in diagnosing LSCC.

Direct low field J-edited diffusional proton NMR spectroscopic measurement of COVID-19 inflammatory biomarkers in human serum.

A JEDI NMR pulse experiment incorporating relaxational, diffusional and J-modulation peak editing has been implemented for a low field (80 MHz proton resonance frequency) spectrometer system to measure quantitatively two recently discovered plasma markers of SARS-CoV-2 infection and general inflammation. JEDI spectra capture a unique signature of two biomarker signals from acetylated glycoproteins (Glyc) and the supramolecular phospholipid composite (SPC) signals that are relatively enhanced by the combination of relaxation, diffusion and J-editing properties of the JEDI experiment that strongly attenuate contributions from the other molecular species in plasma. The SPC/Glyc ratio data were essentially identical in the 600 MHz and 80 MHz spectra obtained (R2 = 0.97) and showed significantly different ratios for control (n = 28) versus SARS-CoV-2 positive patients (n = 29) (p = 5.2 × 10-8 and 3.7 × 10-8 respectively). Simplification of the sample preparation allows for data acquisition in a similar time frame to high field machines (∼4 min) and a high-throughput version with 1 min experiment time could be feasible. These data show that these newly discovered inflammatory biomarkers can be measured effectively on low field NMR instruments that do not not require housing in a complex laboratory environment, thus lowering the barrier to clinical translation of this diagnostic technology.

Click Reaction-Assisted Construction of Antifouling Immunosensors for Electrochemical Detection of Cancer Biomarkers in Human Serum

Click Reaction-Assisted Construction of Antifouling Immunosensors for Electrochemical Detection of Cancer Biomarkers in Human Serum

High Sensitivity Label-Free Quantitative Method for Detecting Tumor Biomarkers in Human Serum by Optical Microfiber Couplers.

Label-free optical fiber immunosensors have attracted widespread attention in recent decades due to their high sensitivity. However, nonspecific adsorption in serum has remained a critical bottleneck in existing label-free fiber optic biosensors, which hinders their widespread use in diagnostics. In addition, individual differences in clinical human serum (HS) negatively impact biosensing results. In this work, the modified serum preadsorption strategy was applied to reduce nonspecific adsorption by forming a saturated antifouling interface on an optical microfiber coupler (OMC). Furthermore, to reduce the effect of the differences between individual HS samples, we proposed a new method where Sigma HS was used as a wavelength shift reference due to being close to clinical serum compared to other serums. Sigma HS was used first to reduce the differences in immune sensors before performing a clinical sample test in which quantitative detection was achieved based on the independent calibration of several sensors with wide dynamic ranges via dissociation processes. The individual differences in 25% HS were corrected by 30% Sigma HS. As a proof of concept, the label-free OMC immune sensor demonstrates good sensitivity and specificity for the detection of carcinoembryonic antigen (CEA) in 25% Sigma HS at different concentrations. The detection limit of CEA reached as low as 34.6 fg/mL (0.475 fM). Additionally, label-free quantitative detection of CEA using this OMC immune sensor was verified experimentally according to the calibration line, and the results agree well with clinical examination detection. To our knowledge, it is the first study to employ an OMC immune sensor in point-of-care label-free quantitative detection for clinical HS.

On-line duplex molecularly imprinted solid-phase extraction for analysis of low-abundant biomarkers in human serum by liquid chromatography-tandem mass spectrometry

In the present work, a pair of molecularly imprinted polymers (MIPs) targeting distinct peptide targets were packed into trap columns and combined for automated duplex analysis of two low abundant small cell lung cancer biomarkers (neuron-specific enolase [NSE] and progastrin-releasing peptide [ProGRP]). Optimization of the on-line molecularly imprinted solid-phase extraction (MISPE) protocol ensured that the MIPs had the necessary affinity and selectivity towards their respective signature peptide targets – NLLGLIEAK (ProGRP) and ELPLYR (NSE) – in serum. Two duplex formats were evaluated: a physical mixture of the two MIPs (1:1 w/w ratio) inside a single trap column, and two separate MIP trap columns connected in series. Both duplex formats enabled the extraction of the peptides from serum. However, the trap columns in series gave superior extraction efficiency (85.8±3.8% and 49.1±6.7% for NLLGLIEAK and ELPLYR, respectively). The optimized protocol showed satisfactory intraday (RSD≤23.4 %) and interday (RSD≤14.6%) precision. Duplex analysis of NSE and ProGRP spiked into digested human serum was linear (R2≥0.98) over the disease range (0.3-30 nM). The estimated limit of detection (LOD) and limit of quantification (LOQ) were 0.11 nM and 0.37 nM, respectively, for NSE, and 0.06 nM and 0.2 nM, respectively, for ProGRP. Both biomarkers were determined at clinically relevant levels. To the best of our knowledge, the present work is the first report of an automated MIP duplex biomarker analysis. It represents a proof of concept for clinically viable duplex analysis of low abundant biomarkers present in human serum or other biofluids.

Fabrication of Biosensing Interface with Monolayers.

Surface modification is recognized as one of the fundamental techniques to fabricate biosensing interfaces. This review focuses on the surface modification of carbon substrates (GC and HOPG) and silica with a close-packed monolayer, in particular. In the cases of carbon substrates, GC and HOPG, it was demonstrated that surface modification of carbon substrates with diazonium derivatives could create a close-packed monolayer similar to the self-assembled monolayer (SAM) formation with mercapto derivatives. Similarly, the potential of trialkoxysilanes to form a close-packed monolayer was evaluated, and modification with a close-packed monolayer tended to occur under milder conditions when the trialkoxysilanes had a longer alkyl chain. In these studies, we synthesized surface modification materials having ferrocene as a redox active moiety to explore features of the modified surfaces by an electrochemical method using cyclic voltammetry, where surface concentrations of immobilized molecules and blocking effect were studied to obtain insight for density leading to a close-packed layer. Based on those findings, fabrication of a biosensing interface on the silica sensing chip of the waveguide-mode sensor was carried out using triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties to immobilize antibodies and to suppress nonspecific adsorption of proteins, respectively. The results demonstrate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has the potential to detect several tens ng/mL of biomarkers in human serum with unlabeled detection method.

Enzyme-Free Colorimetric Immunoassay for Protein Biomarker Enabled by Loading and Disassembly Behaviors of Polydopamine Nanoparticles

In this work, a mussel-inspired polydopamine (PDA) nanoparticle with biocompatible and reactive surface characteristics was desirably incorporated with high loading capacity and pH-induced disassembly-releasing behavior toward Fe(III) ions to develop an enzyme-free colorimetric immunoassay. The catechol functional network of PDA can act as a scaffold to coordinate with large amounts of Fe(III) ions to form the PDA-Fe(III) NPs whose coordination state can be tailored by changing the pH values. In detail, PDA-Fe(III) NPs can be maintained in a disassembled tri-coordinate state under alkaline conditions while the highly loaded Fe(III) ions can be easily released under acid conditions to react with ferrocyanide for the in situ generation of Prussian blue (PB) NPs. The detection sensitivity of prostate-specific antigen (PSA) was significantly improved, owing to the high peroxidase-like activity of PB NPs that triggered excellent catalytic effect by the colorimetric reaction. In addition, favorable linearity was found in the range of 0.0005-20 ng mL-1 with a low detection limit of 0.84 pg mL-1 (S/N = 3). Furthermore, excellent selectivity and reproducibility were exhibited by the developed enzyme-free colorimetric immunoassay. It is believed that this proposed PDA-Fe(III) NP-based enzyme-free colorimetric system will offer a facile and reliable tool for the sensitive detection of PSA and other cancer biomarkers in human serum.

Simultaneous determination of 30 neurologically and metabolically important molecules: A sensitive and selective way to measure tyrosine and tryptophan pathway metabolites and other biomarkers in human serum and cerebrospinal fluid

Concurrent measurement of tyrosine, tryptophan and their metabolites, and other co-factors could help to diagnose and better understand a wide range of metabolic and neurological disorders. The two metabolic pathways are closely related to each other through co-factors, regulator molecules and enzymes. By using high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry, we present a robust, selective and comprehensive method to determine 30 molecules within 20 min using a Waters Atlantis dC18. The method was validated according to the guideline of European Medicines Agency on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all >0.998. Intra-day and inter-day accuracy were between 80-119% and 81-117%, precision 1-19% respectively. The method was applied to measure TYR, TRP and their metabolites, and other neurologically important molecules in human serum and CSF samples. The assay can facilitate the diagnosis and is suitable for determination of reference values in clinical laboratories.