The Construction Of Terahertz Liquid-phase Sensing Chip And Its Preliminary Application

Background:Terahertz(THz)spectroscopy,as an emerging noninvasive,nonionizing and label-free detection technology,could unravel the mystery of low-frequency intermolecular vibrational modes and is especially adaptable for the quantitative detection of biomolecules.Nevertheless,traditional THz spectroscopy could obtain a limited absorption signal of tiny dried molecules,due to the mismatch between the wavelengths of THz waves and the dimensions of molecules resulting in low absorption cross-section of the molecules.Many feasible strategies have been proposed to enhance the interaction between THz waves and molecules via evanescent waves(THz attenuated total reflection,THz-ATR)and localized electric field(THz metamaterials),thus improving the detection sensitivity.However,any characteristic spectroscopic features of the biomolecules are often obscured by the strong THz absorption of liquid water.It is difficult to probe the THz signal of target in physiological concentration in aqueous environments.To weaken the interference of solvent water,most proposed THz sensing strategies require that the tested samples be tediously dried or that the solvent water be replaced with a low-absorption medium.Considering that most biomolecules require an aqueous environment,the abovementioned processes are harmful to the original biomolecular activity and severely limit the application scenarios.Therefore,THz sensing strategies that work in aqueous environments are urgently needed for the selective analysis of physiological concentration of biomolecules in the physiological state,have important practical significance for the promotion and application of THz technology in clinical detection.Responsive hydrogels are three-dimensional hydrophilic and insoluble crosslinked polymeric networks that are extensively hydrated.These networks have attained considerable interest in biosensing and drug delivery by modifying specific recognition elements(such as aptamers,antibodies,characteristic molecules,etc.),which have the ability to specifically capture target molecules and then exhibit obvious swelling and de-swelling behavior,thus leading to profound variations in refractive index,volume,and hydration state.Due to the characteristics of target-induced hydration state change,the THz label-free sensing platform integrated with responsive hydrogel can sensitively probe the target-induced changes in the hydrogel by virtue of the water sensitivity of the THz waves.It may provide a strategy for the specific detection of target molecules in aqueous environments.Aiming at the scarce selectivity and unsatisfactory sensitivity of THz spectroscopy for aqueous biological sample detection,a molecule-specific THz biosensor prepared from a human ?-thrombin(h-TB)-responsive aptamer hydrogel-functionalized metamaterial was successfully developed and the feasibility of the proposed THz biosensor was success-fully verified by simulations and experiments.Thus,the proposed strategy,as a model system,brings to light new avenues for the development of molecule-specific THz biosensors suitable for aqueous environments.Methods:1.The construction of aptamer hydrogel-functionalized THz metamaterial:After optimizing the functionalization method of aptamer hydrogel on the THz metamaterial,the THz biosensor was constructed by the aptamer hydrogel-functionalized THz metamaterial.The THz-ATR platform was used to obtain the THz spectral of the aptamer hydrogel in response to h-TB.And the signal of the THz biosensor was obtained from the optimized self-reference reflection measurement mode.The feasibility of the proposed strategy was investigated experimentally and theoretically.2.Evaluating the sensing performance of THz biosensor:Several critical experimental parameters were optimized including the thickness of the aptamer hydrogel,aptamer concentration and crosslinking density and the reaction kinetics of the THz biosensor were further investigated.Then the sensitivity and specificity were evaluated in the quantitative detection of h-TB.The practical sample assays of THz biosensor in human serum were performed to verify its potential for practical clinical sample detection.Results:1.The extinction coefficient of the aptamer hydrogel,measured by THz attenuated total reflection spectroscopy,was lower than that for PBS and showed an increase after incubating in aqueous h-TB.The self-referenced reflection spectra of the THz biosensors were performed using 3D-finite-difference time-domain(FDTD)simulations and the simulated resonance peak intensity reached a maximum as the thickness of hydrogel increases to 50 ?m.After 100 pM h-TB was applied,the simulated harmonic peak intensity decreased significantly,and the relative change rate was 23.26%.Furthermore,we compared the self-referenced reflection spectra of the THz biosensor after incubation in PBS with/without h-TB(100 pM),and the calculated signal variations of the THz biosensor after exposure to h-TB and PBS were 22.19%and 1.32%,respectively.The average pore size of the porous network structure inside the hydrogel,characterized by SEM,increased significantly after incubation in h-TB.2.The reaction conditions of the THz biosensor are optimized as follows:the thickness of the aptamer hydrogel film is 50 ?m,and the cross-linking density(ratio of acrylamide(Am)/N,N'-Methylenebisacrylamide(MBAA)))is 200:2 and the aptamer concentration is 15?M.The signal variation of optimized THz biosensor nearly reaches a plateau after 60 min.The sensing performance of the optimized THz biosensor was investigated for h-TB detection.The signal variation of the THz biosensor revealed linearity with the logarithm of h-TB concentration from 0.5 pM to 500 pM(R2=0.9842)with a limit of detection of 0.21 pM(S/N=3).The degree of interference(DI)of common substances(including bovine thrombin,hemoglobin and bovine serum albumin)is negligible,and their DIs are less than 5%.Finally,the quantitative detection of h-TB was performed in human serum samples with a limit of detection(LOD)of 0.40 pM,indicating that the proposed THz biosensor also showed excellent sensing performance in actual clinical sample detection.Conclusion:The theoretical basis of proposed strategy,aptamer hydrogel-functionalized THz metamaterial,was clarified experimentally and theoretically for thrombin detection,and a molecular-specific THz biosensor was successfully constructed.The proposed strategy shows excellent sensing performance in the quantitative detection of thrombin,outperforming most label-free sensing strategies.The potential of practical assay has been verified in human serum samples.The proposed strategy with good versatility can brings to light new avenues for improving the specificity for THz biosensing in aqueous environment.