| ObjectiveAs osteoporosis is a worldwide public health problem,early detection,diagnosis and treatment are crucial to the prognosis of patients with osteoporosis.The existing gold standard test has a certain lag,and it is of great clinical importance to establish a method that can monitor osteoporosis quickly and sensitively.In this study,a new method for the detection of type I procollagen amino-terminal peptide(P1NP),a marker of osteoporosis,was developed by combining Surface-enhanced Raman Scattering(SERS)and Lateral Flow Immunoassay(LFIA),enabling rapid and sensitive monitoring of the development of osteoporosis.MethodsThis study is divided into five main parts: the first part provides an understanding of the current status and hot spots of P1 NP research in osteoporosis through bibliometric analysis to guide subsequent studies.The second part is the preparation of high performance magnetic SERS substrates.In this experiment,the magnetic core gold-shell nanoparticles(Fe3O4@Au MNPs)with high SERS performance were prepared by polyethyleneimine(PEI)-mediated seed growth method,and the preparation process of Fe3O4@Au MNPs was characterized by transmission electron microscopy(TEM),ultraviolet-visible spectrophotometer(UV-vis),and zeta potential measurement instrument to verify the material properties.The third part is to establish the detection system of SERS-LFIA based on Fe3O4@Au MNPs.Briefly,the P1 NP capture antibody was coupled to the surface of Fe3O4@Au MNPs by the amino-carboxyl dehydration condensation reaction,and the P1 NP detection antibody was sprayed onto the nitrocellulose membrane(NC membrane)at the position of the test line(T-line)using a scribing instrument;after optimizing the type of nitrocellulose membrane,the composition of the loading solution,and the antibody modified on Fe3O4@Au MNPs and the detection line(T-line),the SERSLFIA detection system based on Fe3O4@Au MNPs was established.In the fourth part,the SERS-LFIA assay system based on Fe3O4@Au MNPs was used for the detection of P1 NP standard samples,and a calibration curve was established to correlate the P1 NP concentration with the SERS signal intensity,and the sensitivity of the assay results was compared with the conventional colloidal gold-based SERS-LFIA;other bone metabolism markers such as osteocalcin and osteoprotegerin were detected under the established assay system to verify the specificity of this experimental method;standard samples with the same P1 NP concentration were detected using five different batches of prepared test strips to verify the reproducibility of the experimental results.In the fifth part,the established assay system was used to quantify P1 NP concentrations in 72 human sera,including 47 serum samples from healthy subjects,20 serum samples with osteopenia and 5 serum samples with osteoporosis,and to verify the clinical feasibility of the assay system based on Fe3O4@Au MNPs with SERS-LFIA by comparing the quantitative results with those of commercial ELISA kits;the results of the three groups of serum samples were analyzed statistically.ResultsFrom the bibliometric results,it can be seen that research papers on P1 NP monitoring of osteoporosis have steadily and consistently increased from 2003 to the present,with China,the United States,and the United Kingdom being the three countries with the highest number of publications,and the University of Sheffield in the United Kingdom being the most authoritative institution in the field of P1 NP monitoring of osteoporosis research,and its Professor Eastell Richard of the Mellanby Musculoskeletal Research Centre being the leader in this research area.From the research hotspots and subject terms,it is clear that P1 NP plays a pivotal role in predicting fragility fractures in osteoporosis,monitoring the efficacy of osteoporosis drugs,and screening for secondary osteoporosis.The results of TEM,zeta potential,and UV-vis verified the feasibility of the PEI-mediated seed growth method and demonstrated the successful preparation of Fe3O4@Au MNPs.Through optimization experiments,it was determined that CN95 membrane with a pore size of 15 μm was used as the nitrocellulose membrane,phosphate buffer containing 1% Tween-20 and 1% bovine serum albumin was used as the loading solution,and 10 μg P1 NP capture antibody was modified on the surface of Fe3O4@Au MNPs,and the scribed concentration of P1 NP detection antibody was 2.0 mg/m L.Under the optimal optimized conditions,the detection sensitivity of the The detection sensitivity of P1 NP standard samples reached 0.03 ng/m L,which is 10 times more sensitive than the conventional colloidal gold SERS-LFIA.The SERS-LFIA assay based on Fe3O4@Au MNPs has good specificity and reproducibility in the detection of P1 NP.Finally,the ability to accurately quantify P1 NP in human serum samples did not differ significantly from ELISA results(p>0.05),and significant differences were found between P1 NP concentrations in healthy populations and those in patients with osteopenia and in patients with osteoporosis(p<0.05),thus allowing the monitoring of the development of osteoporosis and the assessment of bone loss.ConclusionsLiterature metrics confirm the promising application of P1 NP in monitoring the development of osteoporosis.In this experiment,a SERS-LFIA assay system based on Fe3O4@Au MNPs was successfully established,which can detect P1 NP rapidly and sensitively without crosstalk with other bone metabolism markers,and the results are stable and reproducible;the method can accurately quantify P1 NP concentrations in human serum samples without significant differences from ELISA quantification results,and can differentiate between healthy people and patients with osteoporosis.In addition,the assay takes only 40 minutes,is simple to perform and does not require strict environmental conditions,and can be used as a point-of-care testing(POCT)method for early diagnosis of osteoporosis and monitoring of bone loss. |
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