Biosensor-based MicroRNA Detection And Its Forensic Application In Body Fluid Identification

BackgroundIn forensic investigation,body fluids play an important rule in identification of case nature and reconstruction of case scene,once collected and correctly identified.Currently,numerous types of body fluid identification methods have been developed,such as chemical tests,immunological tests,protein catalytic activity tests,spectroscopic methods and microscopy.However,these conventional methods are mostly presumptive,and have deficiencies such as low sensitivity or specificity,easy to be influenced by putrefaction.Therefore,the use of molecular genetics-based approaches,such as messenger RNA(m RNAs),tissue-specific differentially methylated regions(t DMRs),micro RNAs(mi RNAs),have been recently proposed to supplant conventional body fluid identification methods.As a class of endogenous,non-coding,small,single-stranded RNAs,mi RNAs have been confirmed to be differential in expression profile in different types of body fluids.Mi RNAs can tolerance the degradation of putrefaction,owing to its short sequence length.It has been confirmed that mi RNA markers are practically not prone to degradation in samples of venous blood and semen stains stored for 1 year.These features made mi RNAs become a new molecular tool for body fluid identification in forensic practice.The expression profiles of mi RNAs gradually become a potential platform for identification of different body fluids,such as urine,venous blood,menstrual blood,amniotic fluid,semen and vaginal secretions.Mi RNAs are of low abundance(represent only 0.01%)in total RNA samples,resulting in challenge for quantitative analysis.Some conventional methods,including northern blotting,molecular cloning,microarray and reverse transcription polymerase chain reaction(RT-PCR),have deficiencies such as low sensitivity,costly instruments-dependent.In recent years,various biosensor platforms have been developed in multi fields for nucleic acid detection,owing to its advantages of high efficiency,rapid,specific,cost-effective and sensitive.Meanwhile,as a class of short sequence length RNA,mi RNA can be influenced by RNase.Previous studies on the stability of mi RNA were mostly preserved under freezon conditions in liquid.In forensic practice,biological evidences often exist at room temperature or even extreme conditions(such as heat,man-made damage).Besides,long term freezing storage of biomaterials is expensive.Therefore,explore the effect of different preservation conditions on the degradation of mi RNA is necessary.It can provide proposal on long term storage of biomaterials which need further mi RNA detection.Moreover,the law of mi RNA degradation under different preservation conditions is expected to provide theoretical basis for extrapolation of the time of body fluid formation.ObjectiveTo develop a high efficiency,rapid and sensitive method for mi RNA detection based on biosensor strategy,and afford a new platform for identification of body fluid in forensic practice for trace biological evidences.To explore the law of time-dependent mi RNA degradation under different preservation conditions in vitro,providing evidence for preservation condition of biomaterials which need further mi RNA detection and theoretical basis for extrapolation of the time of body fluid spot(mark / trace)formation.MethodsTarget-triggered three-way junction(3-WJ)structure was induced to enhance the specificity.Exponential amplification reaction(EXPAR)coupled with “positive feedback” region in the 3-WJ template was induced to enhance the amplification efficiency.Mi RNAs with different substance forms(dry spot and solution),different solvents(DEPC-treated water and human serum)and different preservation temperatures(4? and 25?)were stored for 1 day,3days,1 week,2 weeks,3 weeks,4 weeks before used,respectively.The concentration of mi RNA with different preservation conditions was detected using the developed method.Results and discussionThe proposed electrochemical strategy shows high sequence specificity and reproducibility with a dynamic response range from 1 f M to 1 n M,and a low detection limit of 0.14 f M.The plot of the response vs.the logarithm of target mi RNA concentration showed a strong linear relationship in the range.The resulting linear equation was ip(?A)= 1.97e-6+1.91e-6 lg Cmi RNA with a correlation coefficient of 0.9996.The target-triggered 3-WJ structure obtained high sequence specificity to distinguish even single-base mismatched mi RNA sequence.The designed “positive feedback” region in the 3-WJ template obtained an increasing signal readout with ~1.5 fold to 3-WJ template without “positive feedback” region.Recovery test performed in 10% human serum sample demonstrated recoveries between 95 % to 104 %.Of the 10 menstrual blood samples,only 2 DPV signal outputs were detected.RT-q PCR detection revealed that the relative expression ratios of target mi RNA in 10 menstrual blood samples were between 0.313227 to 2.071213.Samples with DPV signal outputs were of higher relative expression ratios.The concentration of mi RNA in dry spots did not significantly change with different preservation conditions(p > 0.01).Mi RNA solution made by DEPC-treated water stored at 4 ?,80%-90%(1 day),46%-54%(3 days),26%-32%(1 week),0.04%-0.06%(2 weeks)of the initial amount remained,respectively.While stored at 25 ?,68%-71%(1 day),22%-25%(3 days),0.009%-0.16%(1 week)of the initial amount remained,respectively.Mi RNA solution made by human serum stored at 4 ?,70%-80%(1 day),35%-42%(3 days),0.07%-0.1%(1 week)of the initial amount remained,respectively.While stored at 25 ?,38%-45%(1 day),18%-19%(3 days)of the initial amount remained,respectively.The concentration of mi R-214 in solution decreased exponentially with the prolongation of storage time(P < 0.01).Conclusionwe developed an electrochemical biosensor for mi RNA detection on the basis of target-triggered 3-WJ structure induced polymerase/nicking enzyme isothermal machine with a “positive feedback” region in the 3-WJ template.The proposed strategy could realize ultra-sensitive and specific detection of mi RNA,with remarkable precision and reproducibility.Although only 2 weak DPV outputs were detected in 10 menstrual blood samples,the recovery tests showed that the method was with tolerant to complex samples.Therefore,the proposed strategy still affords a new platform for mi RNA detection and acceptable accuracy for practical sample analysis.Different preservation conditions do not significantly influence the synthetic mi RNA concentration in dry spots within 4 weeks.Dry spots of body fluid is expected to realize long term storage of mi RNA.The concentration of synthetic mi RNA in solutions decreases with the preservation prolonged.The decreasing tendency is extremely obvious in human serum solution stored at 25?.The law of time-dependent change of mi RNA is expected to provide a theoretical basis for the time of body fluid formation in humid environment.