| BackgroundDNA and biochemical markers play an important role in life science and are widely used in clinical diagnosis,environmental monitoring,food safety and agriculture protection.At present,the detection of DNA and biochemical markers still mainly depends on the modern laboratory environment,expensive equipment and professional operators.This limits the application of DNA and biochemical markers detection on site or in resource-limited settings.Point-of-care testing(POCT)is rapid,simple,highly integrated,does not rely on large-scale equipment and professional operators,which has great potential for application in resource-limited settings.So far,the point-of-care testing still has some limitations: First of all,most devices can only implement on one or several steps in a POCT manner,such as nucleic acid extraction or result readout,and cannot completely achieve the “sample-to-answer” detection in the whole process.Secondly,the POCT devices were often designed for one or a few types of DNA targets from limited sample sources,and cannot work as a versatile platform.To sum up,in this study,we constructed two intelligent POCT platforms(iPOCT)for detection of DNA and biochemical markers,and systematically evaluated their clinical application performance.We hope our study could provide some research basis for further POCT development and clinical translation.Objectives1.To construct an ultra-portable and versatile i POCT platform for DNA analysis.2.To evaluate the clinical application performance of the i POCT platform for DNA analysis.3.To construct a sample-to-answer i POCT platform for biochemical markers quantitation.4.To evaluate the clinical application performance of the sample-to-answer i POCT platform for biochemical markers quantitation.Methods1.The 3D printing and microfluidics were used to design an integrated chip including a 3D printed pre-treatment unit and a microfluidic signal amplification unit.A foldable box and a smartphone light path system were designed.A smartphone temperature control system and a smartphone result readout system were developed.The isothermal recombinase polymerase amplification method and gold-silver nanoparticles were integrated to develop an iRPAS(isothermal recombinase polymerase Au-silver,i RPAS system)triple signal amplification system.Whole blood,buccal,urine samples were detected to verify the ability of the POCT platform for detection of different types of gene mutations,urinary bacterial infections and SNPs.Using bacteria infected milk samples,bacteria infected river water samples,and bacteria infected plant leaf samples,we verified the ability of the i POCT platform for detection of food samples,environmental samples,and agriculture samples.2.Different ambient temperatures were simulated to test the detection performance of the smartphone temperature control system in extreme environments and the battery power consumption.The plasmids containing the thalassemia gene mutation were constructed and the sensitivity and limit of detection of the iPOCT platform were determined.By measuring the detection performance of the i-chips placed at room temperature every week,the stability of the i-chip without cold chain storage was analyzed.By comparing the detection results between trained and untrained user groups,the stability among the users was analyzed.The stability among different brands of smartphones was also tested by using different brands of smartphones.By detecting 172 clinical samples,the accuracy,positive predictive value,and negative predictive value for detection of different types of gene mutations,SNPs,and bacterial detection were analyzed.3.The plasma separation module was constructed using lateral chromatography and 3D printing technology.The separation performance of the plasma separation module was observed by the microscope.The detection module was developed by 3D printing.Using different concentrations of the serum chemical control,the stability of the detection module was analyzed.The smartphone ambient light sensor was utilized and the “Light to Concentration” application was designed to record the transmitted light intensity and convert it to the concentration.4.The series dilutions of the serum chemical control were utilized to determine the linear range and the detection sensitivity of total bilirubin and direct bilirubin.Using 10 biochemical markers with similar detection wavelengths in the blood,the specificity of the sample-to-answer platform was analyzed.The recovery rate of the sample-to-answer platform was evaluated by adding the serum chemical control into real plasmas.By detecting 19 clinical blood samples,the quantitative performance of the sample-to-answer platform for total bilirubin and direct bilirubin in clinical blood samples was verified and compared with the clinical chemistry analyzer.The Passing-Bablok regression analysis,the Bland-Altman difference plot analysis and kappa test were utilized to analyze the correlation and consistency with our platform and the clinical chemistry analyzer.Results1.A POCT platform composed of an i-chip and a f-box termed POCKET was constructed using 3D printing and microfluidic.The platform weighs less than 100 grams and is less than 25 cm in length.The i RPAS triple signal amplification system was established and optimized,and the results after iRPAS amplification could be semi-quantified by smartphone.A smartphone temperature control system was successfully constructed,and a thermal insulation bag was designed for cold environments insulation.Strong signals were observed using the POCKET platform to detect various types of gene mutations,SNPs,and bacterial infections.POCKET platform achieved positive results in blood,buccal,urine,milk,river water and leaves samples.2.The time to reach and maintain 37 °C was less than 20 minutes using the smartphone temperature control system at different ambient temperatures(4 °C,15 °C,25 °C,37 °C).Significantly strong signals were also observed at different ambient temperatures.The smartphone battery was available to perform the detection at least four times.The calculated limit of detection of the POCKET platform was 113 copies/m L.No significant changes in results were observed after 10 weeks storage in a 25 °C incubator.No significant differences were observed between the trained and untrained groups and among three different brands of smartphones.The total accuracy of the POCKET platform for clinical sample detection was 97.1%,the AUC of each target were more than 0.950,the specificities were all more than 77.8%,and the positive predictive values were all more than 88.2%.3.Using lateral chromatography and 3D printing,a sample-to-answer quantitative platform composed of a plasma separation module and a detection module was successfully constructed.The total weight of the platform was less than 40 grams,the cost was less than 5 dollars,and the entire detection cycle was less than 5 minutes.The tail of the plasma separation test strip has the best plasma separation performance,where could be used for the plasma transfer.The detection module was stable within 30 minutes of observation time.The “Light to Concentration” smartphone application was successfully designed to read and record the light intensity data from the smartphone ambient light sensor and convert it to the concentration of biochemical markers.4.The linear regression range of total bilirubin was in the range of 1.0 ?M to 30.3 ?M,and the limit of detection was 0.8 ?M.The regression equation of calibration curve was y = 10.86 * ln(x)+ 4.17.The linear regression range of direct bilirubin was 0.7 ?M to 21.3 ?M.The regression equation of calibration curve was y = 19.5 * ln(x)+ 15.00,and the limit of detection was 0.5 ?M.No significant change of the light intensity signals of 10 biochemical markers were observed in total bilirubin and direct bilirubin detections.The average recoveries of total bilirubin and direct bilirubin were higher than 95%.The accuracy of total bilirubin was 89.5%,the positive predictive value was 100.0%,and the negative predictive value was 84.6%.The accuracy of direct bilirubin was 94.7%.The positive predictive value was 90.0% and the negative predictive value was 100.0%.The Passing-Bablok regression analysis,the Bland-Altman difference plot analysis and the kappa test all showed that our platform has a good correlation and consistency with the clinical chemistry analyzer.Conclusions1.We established an ultra-portable and versatile i POCT platform for DNA analysis termed POCKET.Firstly,the platform is ultra-portable from sample preparation to signal amplification to result readout.The POCKET is less than 100 grams and smaller than 25 cm in length.We developed the iRPAS triple signal amplification system and smartphone temperature control system.Smartphone could be used as a temperature controller,a signal detector and a result readout in different ambient temperature environments.Secondly,the POCKET platform is versatile for detecting different types of DNA including various types of gene mutations,SNPs,and bacterial as well as detecting different types of DNA from a variety of sample sources from clinics(blood,buccal,and urine)to food(milk),to environment(river water),and to agriculture(plant leaves).2.The POCKET platform has a stable and good performance under different ambient temperatures.The detection is specific(single-base differentiation),speedy(< 2 hours),stable(> 10 weeks shelf life),user-friendly and compatible with different brands of smartphones.By analyzing and measuring 172 clinical samples,our platform proved its excellent performance for gene mutation,bacterial infection and SNP detection.3.We established a sample-to-answer quantitative platform for biochemical markers quantitation in whole blood.The platform integrated a plasma separation module and a detection module.The plasma separation module enables rapid plasma separation.The detection module enables biochemical reactions without the need of pipetting.Utilizing the smartphone ambient light sensor and the “Light to Concentration”,the reading and recording of the light intensity was realized and the light intensity was converted into the concentration of biochemical marker.The feasibility of the platform was verified by using detection of total bilirubin and direct bilirubin.4.Our platform exhibited remarkable advantages of significant portability(< 40 g),low cost(< $5),rapidity(< 5 min),instrument-free,high sensitivity(< 1 ?M),and accuracy(89.5% for TBil,94.7% for DBil).19 clinical samples were analyzed,the quantitative performance of our platform for clinical samples was highly consistent with the well-established clinical chemistry analyzer. |
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