Organic metal chalcogenide-assisted metabolic molecular diagnosis of central precocious puberty

Metabolic analysis in biofluids based on laser desorption/ionization mass spectrometry (LDI-MS), featuring rapidity, simplicity, small sample volume and high throughput, is expected to be a powerful diagnostic tool. Nevertheless, the signals of most metabolic biomarkers obtained by matrix-assisted LDI-MS are too limited to achieve a highly accurate diagnosis due to serious background interference. To address this issue, nanomaterials have been frequently adopted in LDI-MS as substrates. However, the “trial and error” approach still dominates the development of new substrates. Therefore, rational design of novel LDI-MS substrates showing high desorption/ionization efficiency and no background interference is extremely desired. Herein, four few-layered organic metal chalcogenides (OMCs) were precisely designed and for the first time investigated as substrates in LDI-MS, which allowed a favorable internal energy and charge transfer by changing the functional groups of organic ligands and metal nodes. As a result, the optimized OMC-assisted platform satisfyingly enhanced the mass signal by ≈10 000 fold in detecting typical metabolites and successfully detected different saccharides. In addition, a high accuracy diagnosis of central precocious puberty (CPP) with potential biomarkers of 12 metabolites was realized. This work is not only expected to provide a universal detection tool for large-scale clinical diagnosis, but also provides an idea for the design and selection of LDI-MS substrates.

Synthesis of four OMCs.The OMCs nanosheets were prepared in a similar method by varying the organic ligands and metal ions according to a reported literature with some modifications. [1]ynthesis of Cu(SPh-COOH) nanosheet.CuCl 2 •2H 2 O (12 mg) was dissolved in 200 mL of ethanol and water (1:1, v/v), then 30 mg 4-Mercaptobenzoic acid was added into the solution, stirred at 100 °C for 6 h, and continued to stir for 12 h after cooling down.Finally, the Cu(SPh-COOH) nanosheet was obtained by centrifugation and washed with ethanol and water, respectively.Synthesis of Ag(SPh-COOH) nanosheet.4-Mercaptobenzoic acid (45.45 mg) and AgNO 3 (25 mg) were dissolved in 250 mL of water and the mixed solution was sonicated at 400 W for 10 min.Then the solution was kept at a constant temperature in an oven at 100 °C for 24 h to obtain a white suspension and a white precipitate.The product was collected by centrifugation of the upper white suspension at 10,000 rpm and washed sequentially with ethanol and water, and finally dried under vacuum at 65 °C.Synthesis of Ag(SPh-OH) nanosheet.25 mg AgNO 3 was dissolved in 100 mL of water, then 100 μL 4-hydroxythiophenol was added into the solution.Later, 1 mL of triethylamine was added, stirred at 85 °C for 6 h, and continued to stir for 12 h after cooling down.The Ag(SPh-OH) nanosheet was obtained by centrifugation and washed with ethanol and water, respectively.Synthesis of Ag(SPh-F) nanosheet.20 mg of AgNO 3 was dissolved in 30 mL acetonitrile and 80 μL of 4-Fluorothiophenol was added dropwise to obtain a milky suspension.Next, 0.5 mL of triethylamine was added dropwise and the milky suspension was instantly clarified.Finally, it was stirred at room temperature until silver-white nanosheet was obtained.The Ag(SPh-F) nanosheet was collected by centrifugation and washed with ethanol.
Characterization.Scanning electron microscopy (SEM) images were collected by a FEI Quanta 250 scanning electron microscope.Transmission electron microscopy (TEM) images were taken with a Hitachi HT7700.The powder Xray diffraction (PXRD) patterns were acquired on a X'Pert-Pro MPD (Philips, Holland) power diffractometer.Atomic force microscopy (AFM) images were taken with a Bruker Dimension ICON atomic force microscope.UV-visible absorption spectra of the materials were obtained by UV-Vis spectrum measurements (Agilent Cary 7000).The zeta potential was measured from dynamic light scattering (DLS, Zetasizer NanoZS90, Malvern Instruments Ltd, UK).The photocurrent was carried out in a standard three-electrode system with materials as the working electrode, platinum wire as a counter electrode, Ag/AgCl as a reference electrode, and 0.5 M Na 2 SO 4 as electrolyte solution with 30 μL Nafion in a CHI 660E electrochemistry workstation (ChenHua Instruments, Shanghai, China).

Density function theory (DFT) calculation. The critical desorption step of
[Glc+Na] + from the OMCs surface was the prerequisite of [Glc+Na] + formation and determined whether it could be detected.All calculations were carried out with CP2K package (version 7.1) in the framework of the density functional theory, [3] based on the hybrid Gaussian and plan-wave scheme. [4]Molecular orbitals of the valence electrons were expanded into DZVP-MOLOPT-SR-GTH basis sets, [5] while atomic core electrons are described through Goedecker-Teter-Hutter (GTH) pseudopotentials. [6]A plane-wave density cutoff of 500 Ry was adopted.The long range van der Waals interaction is described by the DFT-D3 approach. [7]All the structures fully relaxed by CP2K with BFGS scheme, and the force convergence criterion was set to 4.5 * 10 -4 hartree/bhor.The multiplicity of the system was tested until converged.The configuration of [Glc+Na] + and the preferential orientation of glucose molecule on the OMCs surface referred to the previous literature. [8]In addition, the desorption energy (E d ) was calculated by the following formula: where E substrate-adsorbate   =   +   -  - represented the energy of whole adsorption model, E substrate and E adsorbate were the energies of substrate and adsorbate.
Sample collection.This study obeyed the Declaration of Helsinki and was approved by the Ethics Committee of Fuzhou Children's Hospital.Serum samples were collected from patients who were diagnosed as central precocious puberty (CCP) according to a clinically strict diagnostic protocol and were not treated before in Fuzhou Children's Hospital (Fuzhou, China).In addition, exclusion criteria were other forms of PP, obesity, chronic diseases, developmental anomalies, medication use, and history of radiotherapy or chemotherapy.Healthy control serum samples were collected from individuals for health examination who were not diagnosed with CPP.The serum samples were stored at -80 °C before use.
Sample Preparation.Serum samples, standard metabolites, and prepared mixtures were dissolved in deionized water, then 1 μL solution spotted on the polish plate and dried at room temperature for subsequent LDI MS detection.With no expensive and time-consuming pretreatments, the serum samples were simply diluted 10-fold for analysis to gather m/z values under 600 Da. 4-MeBP chloride was dissolved in ethanol /water (1:1, v/v) with the concentration of 2 μM.For the matrixes preparation, DHB (10 mg/mL) was dissolved in acetonitrile/ water containing 0.1% TFA (3:7, v/v).CHCA (10 mg/mL) was dissolved in acetonitrile/water containing 0.1% TFA (3:7, v/v).

Mass Spectrometry Analysis. All LDI MS detections were performed on
Autoflex MALDI-TOF/TOF (Bruker) with the Nd:YAG laser (2 kHz, wavelength of 355 nm) and smart beam system.The acquisitions were carried out in positive ion reflector mode and the MS/MS was in LIFT MS/MS positive mode.MS spectra were generated at 1 kHz and 20 kV with the delay time of 250 ns and the number of laser shots of 300.The program was pre-calibrated by standard molecules.All spectra were used directly without any smoothing procedures.The p value was obtained from the t-test of single m/z signal between diseases and controls.

Statistical
analysis.A total of 313 individuals were divided into CPP group (157 serum samples from CPP patients) and control group (156 serum samples from healthy individuals).No significant difference was found in age among controls and CPP patients by one-way ANOVA performed on SPSS (version 19.0, SPSS Inc., Chicago).The raw data of mass spectra was extracted in Flexanalysis 3.4.The model of OPLS-DA based on paretovariance (Par) scaling and the permutation tests with 200 iterations were established by SIMCA (version 14.0, Umetrics, Umeå, Sweden).The ROC curves were established at Metaboanalyst 5.0 (McGill University, Montreal, Canada, https://www.metaboanalyst.ca/).The searching of potential biomarkers was performed with the limitation of 10 ppm in Human Metabolome Database (https://hmdb.ca/).

Figure S15 .
Figure S15.Power analysis of pilot data.The metabolic fingerprinting extracted from 12 samples (6/6 central precocious puberty (CPP)/HC) were analysed to compute the minimum number of samples required with meaningful machine learning.

Figure S16 .
Figure S16.The permutations plot for OPLS-DA of CPP vs HC.

Figure S18 .
Figure S18.MS/MS results of a) serotonin, b) 199.085Da in CPP, c) dopamine, and d) 176.069Da in CPP.Signals of fragment ions as marked were matched with human metabolome database.

Figure S19 .
Figure S19.The enrichment of different sets in serum.

Table S2 .
Identification of the metabolites.