Detection Of Skin Cholesterol Based On Diffuse Reflectance Spectroscopy

Atherosclerotic cardiovascular diseases(ASCVD)is a major health problem in the world.The diagnosis and prevention of ASCVD have attracted more and more attentions.The accumulation of skin cholesterol has been implicated and become a new biomaeker in the progression of ASVCD.Measuring of skin cholesterol levels by diffuse reflectance spectroscopy is an alternative tool for early screening and risk assessment of ASCVD.Due to its non-invasion,rapidity,real time and in site,the technique will probably have widespread applications in future.The thesis will focus on the noninvasive measurement of skin cholesterol based on diffuse reflectance spectroscopy,including the development of reagents required to identify and indicate skin cholesterol specifically,the design of integrated spectrum detection system.And experimental studies were carried out by measuring the pig skin cholesterol in vitro and human skin cholesterol in vivo to verify the feasibility of the method and system.The main contents and achievements of this thesis are as follows:1.The development of reagent required to identify and indicate skin cholesterol specifically and the design of spectrum detection system.It divided into two parts:the first part is the development of reagent required to identify and indicate skin cholesterol specifically based on the "three drop method",including the detection reagent "A-C-B" copolymer and hydrogen donor substrate solution.And the effectiveness of the reagents were verified by detecting the cholesterol in the pig skin in vitro.The second part is the design of the spectrum detection system,including hardware and software parts.The hardware part comprises a light source unit,an optical transmission unit,a signal detection unit,a reaction chamber unit and so on.The software section contains a control analysis program for skin cholesterol spectrum measurements.In addition,the shell design,accessories design were also carried out and the entire system design and development were completed finally.2.The performance test of spectrum detection system and the research and correction of factors influencing the measurement.Firstly,the performance of the spectrum detection system designed was test,including light,transmission optical path and micro spectrometer.The reproducibility of the system was verified by the diffuse reflectance standard board and deionized water similar to the object to be tested.Secondly,the factors influencing the system measurement were studied and optimized,including the influence of contact pressure between the system and tissue,HRP enzyme activity,system drift and skin background.Thirdly,the diffuse reflectance spectrum was collected by the developed spectrum detection system,the diffuse reflection absorbance A was calculate and the characteristic wavelength and characteristic band were determined.Finally,according to the combination of cholesterol and the detection reagent "A-C-B" copolymer in an equimolar ratio,concentration gradient of skin cholesterol was simulated by the concentration gradient of the detection reagent,and the diffuse reflection absorbance A was calculated and correlated with the "A-C-B" copolymer concentration.The results showed that a linear increase and significant positive correlation existed between the diffuse reflection absorbance A at the characteristic wavelength 652 nm,the integral intensity of the diffuse reflection absorbance A in the characteristic wavelength bands 620-650 nm with the "A-C-B" copolymer concentration.3.Measuring the pig skin cholesterol in vitro by the spectrum detection systemFirstly,Pig skin samples in vitro containing concentration gradient of cholesterol were obtained by extraction.The extraction steps and conditions were determined.Then,the cholesterol in the extracted samples were measured by the spectrum detection system and analyzed linearly with the extraction time.The results showed the diffuse reflection absorbance A at the characteristic wavelength 652 nm and the integral intensity of the diffuse reflection absorbance A in the characteristic wavelength band 620-650 nm can distinguish and quantify the concentration gradient of cholesterol in the sample,and they were linearly related to the extraction time.4.Measuring the body skin cholesterol in vivo noninvasively by spectrum detection system.A total of 31 subjects were enrolled.The skin cholesterol of subjects were collected using the designed spectrum detection system,and blood cholesterol of the subjects were also gained.Linear fitting and correlation analysis were used to analyse the relationship between skin cholesterol and blood cholesterol,including TC,TG,HDL-C,LDL-C,VLDL-C and other risk indicators of ASCVD derived from these five indicators.The results showed that the growing trend of skin cholesterol was consistent with TC and LDL-C,and significant positive correlations were presented between them with the partial correlation coefficient r were 0.813(P<0.01)and 0.759(P<0.01)after controlling the factors of age,gender and BMI.There was no significant relationship between skin cholesterol and TG,HDL-C and VLDL-C.There were significant positive correlations between skin cholesterol and non-HDL-C,TC/HDL-C,LDL-C/HDL-C and AI,and the partial correlation coefficient r were 0.769(P<0.01),0.674(P<0.01),0.746(P<0.01)and 0.674(P<0.01)after controlling the factors of age,gender and BMI.When combination analysis of skin cholesterol and any two reference indicators were done,skin cholesterol also showed an increasing trend with the growth of reference indicators.Finally,the subjects were divided into normal,low-risk,and high-risk subjects based on the reference criteria mentioned above in the risk assessment of ASCVD.The skin cholesterol between the three groups of subjects had significant differences(one-way ANOVA values were greater than 1,P<0.01),which indicated that skin cholesterol can be used to differentiate the subjects with different degrees of risk of ASCVD.The above studies showed that noninvasive detection of skin cholesterol based on diffuse reflectance spectroscopy would contribute to the early screening and risk assessment of ASCVD,and also do a great favor to the prevention and treatment of ASCVD.