Highly Sensitive And Selective Electrochemical Biosensors For Thrombin And Circulating Tumor Cells

Proteins are the fundamental components of life,which play important role in the life activities.Circulating tumor cells?CTCs?are the main promoters of cancer metastasis,which can be considered as the crucial indicators of metastases.They are the important biomarkers which related to different diseases.Therefore,the detection of disease-related markers,proteins and CTCs,can provide significant information for the disease diagnosis,clinical treatment and prognosis evaluation.However,the biological samples are extremely complex and the concentrations of biomarkers are very low,thus proposing great challenges for the detection of disease markers.Electrochemical biosensor can convert specific biochemical signal into detectable electrochemical signal,which has lots of attractive advantages such as high sensitivity,good selectivity,portable instrument,rapid response and low cost.However,the traditional electrochemical biosensor is still unable to meet the demands of highly sensitive detection of low content disease marker.Therefore,we are aiming to develop highly selective electrochemical biosensor for the sensitive detection of thrombin and CTCs by using aptamers/antibodies with high affinity and specificity as recognition elements.Dual recognition and multivalent binding strategies are adopted for the improvement of the specificity of biosensor.Enzyme,nanomaterials,strand displacement reaction and DNA selfassembly are used as signal amplification approaches to improving sensitivity of biosensor.The experimental results show that the constructed electrochemical biosensors have high sensitivity and selectivity for the detection of disease markers?thrombin and CTCs?.And the thrombin and CTCs spiked in diluted serum or whole blood can be detected by the proposed electrochemical biosensors,suggesting the great potential of the biosensors in clinical samples.The studied contents are mainly as follows:1.Target-programmed and autonomous proximity binding aptasensor for amplified electronic detection of thrombinThe development of sensitive and simple approaches capable of monitoring trace amounts of protein biomarkers is appealing for disease diagnosis and treatment.Towards this end,we have developed an electrochemical sensing platform for sensitive and simple detection of protein biomarkers by using thrombin as the model target molecules via a target-programmed proximity binding amplification approach.The binding of thrombin to the aptamer sequences in the partial dsDNA duplex probes induces the release of the ssDNA trigger strands,which catalyze subsequent assembly formation of many methylene blue?MB?-tagged proximate DNA motifs with the presence of the DNA fuel strands through cascaded toehold-mediated strand displacement reactions.Due to the proximity-binding effect,these MB-tagged proximate DNA motifs anneal with the capture probes on the sensor surface with significantly enhanced stability against the corresponding single component counterpart,thereby pulling the MB tags close to the sensor surface and generating substantially amplified signal responses for sensitive determination of thrombin down to 23.6 pmol L^-1.In addition,such aptasensor can specifically discriminate thrombin from other interference proteins,and can also be utilized to monitor thrombin in diluted serum samples,demonstrating its great potential for sensitive determination of proteins for early disease diagnosis.2.Aptamer/protein proximity binding-triggered molecular machine for amplified electrochemical sensing of thrombinThe development of convenient and sensitive methods without involving any enzymes or complex nanomaterials for the monitoring of proteins is of great significance in disease diagnostics.In this work,we describe the validation of a new aptamer/protein proximity binding-triggered molecular machinery amplification strategy for sensitive electrochemical assay of thrombin in complex serum samples.The sensing interface is prepared by self-assembly of three-stranded DNA complexes on the gold electrode.The association of two distinct functional aptamers with different sites of thrombin triggers proximity binding-induced displacement of one of the short singlestranded DNAs?ssDNAs?from the surface-immobilized three-stranded DNA complexes,exposing a prelocked toehold domain to hybridize with a methylene blue?MB?-tagged fuel ssDNA strand?MB-DNA?.Subsequent toehold-mediated strand displacement by the MB-DNA leads to the release and recycling of the aptamer/protein complexes and the function of the molecular machine.Eventually,a large number of MB-DNA strands are captured by the sensor surface,generating drastically amplified electrochemical responses from the MB tags for sensitive detection of thrombin with a detection limit of 1.7 pmol L^-1.Our signal amplified sensor is completely enzyme-free.Such sensor also has a high specificity for thrombin assay in serum samples.By changing the affinity probe pairs,the developed sensor can be readily expanded as a more general platform for sensitive detection of different types of proteins.3.Proximity binding and metal ion-dependent DNAzyme cyclic amplification-integrated aptasensor for label-free and sensitive electrochemical detection of thrombinThrombin plays important roles for the diagnosis of neurodegenerative and cardiovascular diseases.By integrating proximity binding-induced strand displacement and metal ion-dependent DNAzyme recycling amplification,we demonstrate here the development of a simple and sensitive strategy for the detection of thrombin in human serums.The binding of the two distinct aptamers to the thrombin targets increases the local concentration of the aptamers and facilitates the release of the enzymatic sequences through proximity binding-induced strand displacement.The liberated enzymatic sequences further hybridize with the G-quadruplex containing and hairpin-structured substrate sequences on the sensor electrode to form the metal-ion dependent DNAzymes.Subsequently,the metal ions catalyze the cleavage of the substrate sequences to unlock the G-quadruplex forming sequences and to release the enzymatic sequences to trigger another cleavage cycle.Such metal ion-dependent DNAzyme recycling amplification leads to the formation of many active G-quadruplex forming sequences,which associate with hemin to form G-quadruplex/hemin complexes on the electrode surface.Direct electron transfer of hemin to the electrode during the potential scan can thus generate significantly amplified current for sensitive detection of thrombin at the low picomolar level.The work demonstrated here can thus offer new opportunities for the development of convenient signal amplification strategies for detecting various protein targets.4.Highly specific and sensitive point-of-care detection of rare circulating tumor cells in whole blood via a dual recognition strategyDespite the fact that the identification and detection of circulating tumor cells?CTCs?plays a critical role in cancer monitoring and diagnosis,it remains a major challenge to isolate and detect these cells,due to their extreme scarcity in peripheral blood.In this work,by coupling a dual recognition strategy and the commercial personal glucose meter,we established a point-of-care approach for detecting rare CTCs in whole blood with high sensitivity and selectivity.The antibody-conjugated magnetic beads lead to the capture and isolation of the CTCs while the enzyme-and second antibody-modified microspheres yield the signal for detection.Because of the dual recognition format,the developed method is highly selective,and a low detection limit of 7 cells can be realized as well,owing to the great signal amplification through the enzyme-loaded microbead labels.More importantly,the detection of CTCs in whole blood can be achieved in a point-of-care fashion with the using of the glucose meter transducer,offering our method a convenient and attractive alternative to traditional biopsy for the diagnosis of various cancers.5.In situ-generated multivalent aptamer network for highly efficient capture and sensitive electrochemical detection of circulating tumor cells in whole bloodMonitoring circulating tumor cells?CTCs?in human blood can offer useful information on convenient metastasis diagnosis,prognosis and treatment of cancers.However,it remains a substantial challenge to detect CTCs because of their particular scarcity in complex peripheral blood.Herein,we describe an in situ-generated multivalent aptamer network-modified electrode interface for efficiently capturing and sensitively detecting CTCs in whole blood by electrochemistry.Such an interface was fabricated via rolling circle amplification extension of the electrode-immobilized primer/circular DNA complexes for the yield of long ssDNA strands with many repeated aptamer segments,which could achieve efficient capture of rare CTCs in a multivalent cooperative manner.The antibody and horseradish peroxidase-functionalized gold nanoparticles further specifically associated with the surface-bound CTCs and generated electro-catalytic amplification of current outputs for highly sensitive detection of CTCs with an attractive detection limit of 5 cells.Besides,the multivalent aptamer network interface could successfully distinguish the target cells from other control cells and achieve CTC detection in whole blood,demonstrating its promising potential for monitoring different rare CTCs in human blood.