Novel Homogeneous Electrochemical Biosensing Strategies For Biomarkers Detection

Cancer,known as a life-threatening disease,causes millions of people to die every year due to its high metastatic diffusion capacity and fatality rate,and it significantly affects human health and public safety.Early diagnosis of cancer,which may offer valuable information on enhancing therapeutic efficiency and play a critical role in improving survival rate of cancer patients,is in great demand.But early diagnosis of cancer is still a great challenge.Some traditional techniques,including X-ray,CT,and B ultrasound concentrating on morphological changes of tissues,cannot meet this demand,and they result in missing the best times for cancer therapy.With the development of biological and medical techniques,biomarkers appeared and served as important indices for risk assessment and early cancer detection.And it has been proved that their appearance often precedes cell or histomorphological or biological changes.Therefore,the establishment of a sensitive and selective sensing platform based on disease markers is expected to realize early accurate diagnosis and treatment of diseases.Biosensors,as a new and high-tech technology with the mutual penetration and integration of multiple disciplines,have played an irreplaceable role in the detection of cancer markers due to their good specificity,low detection limit and fast analysis speed,and the high-sensitive detection of target objects in complex samples.In recent years,great efforts have been devoted to developing novel biosensing techniques for cancer early diagnosis by identifying the abnormal expression of biomarkers in biological samples.Among these techniques,the homogeneous electrochemical sensing strategy is regarded as an ideal tool due to its immobilization-free characteristic which not only simplifies the operation procedure and lowers the detection cost but also avoids the steric hindrance effect to strengthen the recognition and response efficiency.However,it is worth noting that these reported homogeneous electrochemical techniques have several of the following drawbacks.First,most of the previously reported homogeneous electrochemical strategies suffered from complicated design ideas and long reaction time,making the development of homogeneous electrochemical biosensors more difficult.Second,most of the previously reported homogeneous electrochemical strategies only have single signal output to sense biosensors.It is easy to be influenced by the environment condition,making the diagnosis results inaccurate and subsequent limiting the development of homogeneous electrochemical biosensors.Third,all the homogeneous electrochemical biosensors commit themselves to single tumor biomarker detection,easily resulting in false positive diagnosis because the expression level of tumor biomarkers can also be influenced by other factors（inflammation and infection）.For the sake of addressing the issues above,we have developed a series of novel homogeneous electrochemical strategy for sensitive detection of multiple tumor biomarkers.（1）A simple,rapid,and sensitive homogeneous electrochemical bleomycin（BLM）bioassay has been successfully developed through the target-induced specific/efficient cleavage reaction.In this work,we designed signal probes,denoted as MB-DNA,contains both methylene blue（MB）and target recognizable sequences.When BLM is not present,MB-DNA could not be recognized/cleaved,and still maintain its high negative charge density,subsequently leading to strong repulsive force between MB-DNA and ITO electrode.Owing to the diffusion effect,which is dependent on the repulsive force,weak electrochemical signal is observed for the MB-DNA probe.Nevertheless,upon the addition of BLM,BLM could react with Fe²⁺to form the BLM-Fe²⁺complex.Owing to the specific/efficient cleavage reaction of MB-DNA by BLM-Fe²⁺complex,a large number of two-mer MB-lined oligonucleotide fragments（MB-DNA-1）are released,of which negatively charge density remarkably reduces compared with that of MB-DNA.And MB-lined oligonucleotide fragments were easily diffused to the electrodes.As a result,an obvious enhancement in electrochemical signal is determined.Thus,a turn on homogeneous electrochemical method for BLM is really achieved,and exhibits high sensitivity of 33 pM,and the shortest response time of 20min.Comparing with other sensing strategies for BLM,this proposed electrochemical platform is just consisted of one DNA probe alone,and affords a really rapid and sensitive strategy for BLM analysis.（2）A truly RHE biosensor for miRNA-182 sensitive biosensing based on the unique diffusion/intercalation characters of electroactive dyes without the need to electrode modification or material preparation has been successfully developed.When target miRNA-182 is introduced into the sensing system,the hybridization recognition of Fc-H1 by miRNA-182 impels the digestion by Exo III,thus contributing to the release of ssDNA trigger s1 and the production of Fc-mNs,which significantly enhances the current response of Fc due to the increased diffusion to the ITO electrode.The released s1 initiated hybridization chain reaction（HCR）of H2 and H3 to form a long chain of dsDNA with a single-strand overhang on the 3′and 5′ends bringing them close to each other to form G-quadruplexes in the presence of K⁺.Greater numbers of MB molecules are trapped and prevented from moving to the ITO electrode,resulting in an obvious decrease in the DPV signal of MB.When target miRNA-182 was absent in solution,three probes of Fc-H1,H2,and H3 maintain their hairpin configurations unchanged.The Fc-H1 affords a relatively low DPV signal and large numbers of MB molecules are free in the sensing system and enjoy a high current response of MB.Thus,the opposite current changes of MB and Fc in the presence of miRNA-182 reveal that the exceptional miRNA sensing with high sensitivity and good selectivity was readily realized on the account of I_Fc/I_MB value change.More importantly,it exhibited fantastic accuracy and reliability for target miRNA detection in diluted serum obtained from glioma patients.Furthermore,this work revealed that such proposed strategy may be more suitable for future development of novel ratiometric homogeneous electrochemical biosensor because of the occurrence of all reactions in the same solution and no involvement of electrode modification/material preparation.Thus,this study was expected to provide a new thinking way for development of ratiometric homogeneous electrochemical biosensors for miRNA-related diseases diagnosis.（3）A novel homogeneous electrochemical biosensor for simultaneous detection of multiple tumor biomarkers based on the functionalized MOFs.The functionalized MOFs were prepared by using porous UIO-66-NH₂ as nanocontainer to load electroactive dyes（MB and TMB）and dsDNA as a gatekeeper to cap MOFs to produce MB@UIO and TMB@UIO,respectively,both of which could be used as homogeneous electrochemical biosensors for let-7a and miRNA-21 simultaneous detection.In the absence of target analytes,the dsDNA on MOFs prevented the release of MB and TMB.None of MB and TMB could diffuse to the electrode.As such,the peak currents of MB and TMB were ultralow.Upon the addition of the two biomarkers,both MB and TMB were allowed to simultaneously release into the sensing system,two stronger signals are recorded,and dependent on target miRNA concentrations.Thus,simultaneous detection of let-7a and miRNA-21 is achieved,with detection limits down to 3.6 and8.2 fM,respectively.Moreover,the proposed biosensor has also been successfully applied for simultaneous detection of target miRNAs spiked in serum samples.Therefore,the proposed strategy was expected to provide more information for early and accurate cancer diagnosis and was a useful application in disease diagnosis and clinical biomedicine.（4）A one-step strategy for the synthesis of methylene blue-encapsulated ZIF-90（MB@ZIF-90）with high loading,unique dual-signal property,exceptional recognitional capability,and good stability was developed for label-free,enzyme-free,and ultrasensitive detection of adenosine triphosphate（ATP）by integration of fluorescent and homogeneous electrochemical techniques.When ATP was absent,MB@ZIF-90 maintained its structure and morphology minor variation,and the unspoiled ZIF-90 prohibited the release of MB.As a result,no or only small MB was observed in the supernatant,and the detected fluorescent and electrochemical signals were very low.Whereas,upon the addition of ATP,MB@ZIF-90 decomposed due to the emulative coordination of ATP and 2-ICA on Zn²⁺,and meanwhile,MB was efficiently released from ZIF-90 into solution.A large diversity of MB was determined in supernatant,and subsequently contributed to both high FL intensity and DPV current.Considering both the increased signals,MB@ZIF-90-based dual-signal biosensor for label-free,enzyme-free and ultrasensitive detection of ATP was developed based on the integration of fluorescent and homogeneous electrochemical techniques.Moreover,the synthetic strategy is widely applicable to other functional molecules for fabricating multifunctional materials for high-performance biosensors development.This work opens a path to explore biosensing application of MOFs,and also provides a simple approach to develop dual-signal biosensors for more accurate and reliable biosensing.