Sepsis is a systemic inflammatory response syndrome(SIRS)caused by pathogenic microorganisms invading the human body,which is a common disease in ICU,the mortality rate is as high as 30% to 50%.If sepsis patients are not correctly diagnosed and treated in time,it will not only significantly prolong their hospitalization time,lead to the abuse of antibiotics,but also increase medical costs.Therefore,accurate early diagnosis and timely treatment are of great significance to the prognosis of sepsis patients.Interleukin-6(IL-6)and Procalcitonin(PCT)as biomarkers for sepsis emerging in different periods of time have differences in response time,sensitivity and specificity,and developing a combined test on the basis of single index test can improve the diagnosis efficiency of sepsis.Therefore,it is necessary to establish a single-and dual-index combined detection method with simple operation,high sensitivity,and rapid quantification.It not only shortens the detection time,realizes "one sample gets two detection values",but also facilitates early diagnosis and subsequent treatment.In this study,3% PBS solution was used as the matrix sample.Au NPs were prepared by reduction of chloroauric acid using trisodium citrate as reducing agent,and Raman reporter molecules were embedded in the nanogap between Au core and Ag shell to form a large number of "hot spots",resulting in the enhancement of the local electromagnetic field and producing the SERS enhancement effect.Using the double-antibody sandwich principle,the tracer antibody coupled to the SERS substrate,the antigen and the capture antibody coupled to biotin can generate a classic "sandwich" double-antibody sandwich structure through an immune reaction.Because of the specific reaction between biotin and streptavidin,the double antibody sandwich structure was enriched by streptavidin modified magnetic beads,and then the magnetic beads were enriched with magnets and the supernatant was discarded.finally,the quantitative detection of biomarkers was realized by collecting Raman signals on the magnetic beads.The results showed that:(1)Surface-enhanced Raman spectroscopy detects IL-6(0.0~1000.0 pg/mL,r=0.9997),the limits of detection was 1.6 pg/mL,the spiked recovery was in the range of 93.9%~99.1%,within the batch of three batches of reagents both inter-assay and intra-assay coefficient of variation(CV)were <20%,the precision and accuracy were in accordance with the detection requirements.there was no cross-reactivity with procalcitonin(20 ng/mL)and Creactive protein(CRP,100 μg/mL),showing good specificity;except for bilirubin(2 mg/mL)and hemoglobin(10 mg/mL),there was no interference with other common interfering substances in serum,showing good anti-interference;57 cases of clinical serum samples were detected simultaneously with chemiluminescence method,the correlation was good(r=0.9896,P<0.01).There was no significant difference between the two methods(t=0.985,P=0.329 >0.05);(2)Surface-enhanced Raman spectroscopy detects PCT(0.0-20.0 ng/mL,r= 0.9998),the limits of detection was 0.012 ng/mL,the spiked recovery was in the range of94.5%~102.9%.both inter-assay and intra-assay CV were < 20%,the precision and accuracy were in accordance with the detection requirements.there was no cross-reactivity with IL-6(1000 pg/mL)and CRP(100 μg/mL),showing good specificity;except for bilirubin(2 mg/mL)and hemoglobin(10 mg/mL),there was no interference with other common interfering substances in the serum,showing good anti-interference;Simultaneously with the industry standard immunochromatography method,59 clinical serum samples were tested at the same time,the correlation was good(r=0.9876,P<0.01).There was no significant difference between the two methods(t=-1.786,P=0.079>0.05);(3)Surface enhanced Raman spectroscopy was used for the combined detection of IL-6(0.0~1000.0 pg/mL,r=0.9992)and PCT(0.0~20.0 ng/mL,r =0.9988),the limits of detection dual index detection were better than single index detection,the limits of detection were 0.54 pg/mL and 0.042 ng/mL,respectively.the spiked recovery was in the range of89.8%~104.2%.both inter-assay and intra-assay CV were < 20%;The results were compared with those obtained for CRP(100 μg/mL)showed no cross reactivity and showed good specificity.Both common interfering substances and common anticoagulants in serum showed little interference with the test results.Using surface-enhanced Raman scattering combined detection,the detection value of IL-6 was compared with 55 clinical serum samples with a chemiluminescence value,the correlation was good(r=0.9712,p<0.01).There was no significant difference between the two methods(t =0.937,p=0.353>0.05);PCT detection value was compared with 59 clinical serum samples determined by immunochromatography,the correlation was good(r=0.9876,p<0.01),There was no significant difference between the two methods(t=-1.786,p=0.079>0.05).The precision and accuracy of the joint detection method meet the requirements of serum sample analysis,and has good selectivity.Compared with the industry standard method,it is more rapid,accurate and sensitive.In this study,the established detection method can quickly and accurately quantitatively detect sepsis biomarkers IL-6 and PCT,and complete dual-index combined detection at the same time.Its operation is simple,the detection time is short,and no professional is required operation,and the entire detection process is green,environmentally friendly,and pollutionfree,and initially realized the ultra-sensitive detection of the biomarker IL-6,which provides a practical basis for early diagnosis of sepsis and bedside monitoring,and lays a foundation for application.It provides a case reference for precise medical analysis of disease biomarkers. |