| Reliable detection of trace biomarker in the early stage of diseases is of great significance for the reduction of disease morbidity and mortality,and circumvention of long-term ineffective treatments and side-effects.On this account,development of methods for rapid and sensitive detection of biomarker has attracted great attention in the fields of clinical diagnosis,biological research and medical therapy.Up to now,various types of biosensors have been developed continually for determination of bi-omarkers.Among them,electrochemical biosensors,relying on their inherent ad-vantages of miniaturization,rapid response,high sensitivity,low cost and convenient operation,hold great potential in the development of point-of-care diagnostic devices In practical clinical diagnosis,however,the complexity of real biological samples usually interferes the accuracy of the biomarker detection.Therefore,it is imperative to develop robust analytical devices with anti-interference performance as well as high sensitivity to meet the practical demands of biomarker detectionRecently,ratiometric electrochemical biosensors,which integrate two electro-chemical sensing principles onto the same platform and use the ratio of the two sig-nals as the final output signal,have been attracting considerable research interest Compared with single-signaling biosensor with ’signal-on’ or ’signal-off’ mode,ra-tiometric electrochemical biosensor with dual-signaling mode provides built-in cor-rection and the circumvention of signal fluctuation caused by complex experimental conditions through their self-referencing capability,11,12 thus exhibits enhanced re-producibility and anti-interference ability.There is no doubt that these preeminent performances endow ratiometric electrochemical biosensor with great potential for clinical application to access reliable and convincing results.According to the mecha-nism of the ratiometric electrochemical biosensor in the on-off mode,amplification of the signal change of any process can effectively augment the ratio of increased signal to decreased signal,thereby improving the sensitivity of the sensor.Hence we have constructed a ultrasensitive ratiometric biosensor for the detection of two very com-mon cancer markers.The specific contents are as follows:1.Bi-directionally amplified ratiometric electrochemical aptasensor based on RecJf exonuclease and bioconjugate probe for ultrasensitive detection of al-pha-fetoprotein(AFP).AFP is one of the most common hepatocellular carcinoma biomarkers.The aptasensor was fabricated by attaching ferrocene-labeled capture probe(Fc-CP)onto electrode surface through the hybridization with thiolated DNA,.The recognition of AFP caused the disassociation of Fc-CP from the DNA1/Fc-CP du-plex,and further triggered the cleavage process of RecJf exonuclease and recycling of AFP.As a result,numerous Fc labels were away from the electrode surface,accompa-nied by plenty exposed DNA1 on the electrode surface for the hybridization with methylene blue(MB)labeled bio-bar-coded gold nanoparticle probe(MB-DNA-AuNP).This probe contained vast MB molecules and resulted in en-hanced MB signal.This bi-directional amplification mechanism significantly ampli-fied the decrease of Fc signal and the increase of MB signal,finally achieving a wide detection range of seven orders of magnitude and an ultralow detection limit down to 269.4 ag/mL for AFP detection.The anti-interference property of the sensor was also verified by analyzing clinical human serum samples.This aptasensor is promising for the development of reliable electrochemical devices for point-of-care diagnosis.2.Bi-directionally amplified ratiometric electrochemical biosensor based on DSN enzyme and bioconjugated probe for detection of miRNA-21.MiRNA,a class of small and noncoding single-stranded RNA with an approximately length of 22 nu-cleotides,plays a significant role in multiple biological processes such as cell differ-entiation,apoptosis,tumor formation and virus replication.Therefore,miRNA is con-sidered a new class of biomarkers for early diagnosis and prognosis of diseases.First,the terminal thiolated,ferrocene-labeled hairpin probe(Fc-HP)was anchored onto the gold electrode surface.When miRNA-21 was introduced,the probe will recognize miRNA-21 and form a DNA-RNA duplex.Relying on the characteristics of DSN en-zyme to hydrolyze the DNA in the double strand,the target miRNA-21 was released for the Fc-HP circular recognition.The remaining Fc-HP hydrolyzed by DSN enzyme on the gold electrode could bind to the pre-synthesized MB-DNA-AuNP bioconjugate probe,which is contained in a large number of MB molecules.Eventually,the Fc electrochemical signal is rapidly reduced and the MB signal is greatly increased,so that the ratio of the Fc to MB respond signal as the ratio output signal is greatly en-hanced,thereby greatly improving the sensitivity of the sensor.Finally,it was verified that the developed Bi-directionally amplified technology can be excellent for detect-ing protein and nucleic acid,two common types of cancer markers,so that it has great application potential in early diagnosis of diseases. |
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