| Therapeutic drug monitoring(TDM)refers to the periodic detection of drug concentration in patient’s body fluids,such as blood,etc.with analytical techniques.Combined with pharmacodynamics and pharmacokinetics,individualized drug administration regimen can be realized with TDM.Thus,it can improve the efficacy of drugs and avoid or reduce the toxic side effects,so as to achieve the purpose of safe and efficient treatment.However,compared with developed countries,about 100 kinds of drugs can be detected,our country is still in the slow development stage,only less than 20 kinds of drugs are routinely monitored.At present,chromatography and immunoassay are the two most widely used techniques in TDM,but they are limited by long time and high cost.Moreover,TDM is mainly carried out in some large hospitals.Therefore,it is of great significance to improve the detection rate and reduce the detection cost for the promotion of TDM.As an important clinical drugs,methotrexate(MTX)has good curative effect on rheumatoid arthritis and a variety of malignant tumors.However,the total body clearance of MTX is highly variable between individuals.Moreover,even a low dose of MTX has side effects,high-dose MTX poses some life-threatening side effects.Measuring MTX in patient blood samples is believed to be important for safe medication and personalized medicine.After the start of the infusion,delayed elimination-induced MTX plasma levels ≥ 50 μM at 24 h,≥ 5 μM at 48 h,≥ 0.2 μM at72 h means the risk of excessive toxicity.Clinical methods for MTX TDM are high performance liquid chromatography(HPLC)and enzyme-linked immune sorbent assay(ELISA).While,HPLC suffers from the high costs of equipment and professional technicians,and ELISA often requires high time cost.Aptamers are single-stranded functional nucleic acids screened by systematic evolution of ligands by exponential enrichment(SELEX).As molecular probes,aptamers can recognize targets with high specificity and affinity,and have been widely used for developing sensors.In this paper,aptamers with high affinity and specificity to MTX were obtained through rigorous in vitro screening and reasonable sequence optimization procedure.Aptasensors HMX38-3,HMX-SNA and HMX-c BCas were designed to rapidly detect early metabolic delay of MTX.Utilizing primer exchange reaction(PER)technology,highly sensitive aptasensor(Apt-PER)were designed for the efficient detection of late MTX metabolic delay.The detailed research subjects are as follows:1.Screening of MTX aptamerAptamer HMX72 was selected using MTX as positive target and folic acid(FA)as negative target.Upon characterization and optimization,aptamers HMX38 and HMX32 with high affinity and specificity were obtained.Further characterization indicated that free HMX38 has a mixed parallel G quadruplex structure,which was changed to parallel G quadruplex upon the binding of MTX,and MTX was compelled to rigidity upon binding.Aptamers such as HMX38 not only have high specificity and affinity which are similar to antibodies,but also have many advantages such as easy synthesis,easy modification,strong programmable,etc.,providing a new tool for the development of rapid and convenient clinical/home MTX detection sensor.2.Design of aptamer-based strand displacement sensor for specific detection of MTXFluorescence molecules were attached to the 5’ end of HMX38,fluorescence quenching molecules were attached to the 3’ end of its complementary strand.Under the effect of fluorescence resonance energy transfer(FRET),the fluorescence molecules were quenched,which can be restored by the binding of MTX.Under this guidance,intermolecular competition based aptasensor HMX38-3 was developed.In50% serum,MTX can be detected within 5 minutes with high specificity using HMX38-3,with a linear detection range of 0.5 ～ 10 μM and a detection limit of 0.18μM,corresponding to 0.36 μM in 100% serum,which indicated the great potential of accurate and efficient early MTX excretion delay detection with HMX38-3.Compared with traditional MTX detection technologies such as ELISA,HPLC and LC-MS,HMX38-3 greatly reduced the detection time and cost,simplified the sample pretreatment process,and provided a new strategy for the rapid and efficient detection of MTX.3.Construction of gold nano-stars based Fluorescence-SERS dual signal aptasensor for specific MTX detectionSurface-enhanced Raman scattering(SERS)and fluorescence quenching effect based on gold nano-stars(GNSs)have been widely used in the development of sensors.For satisfying the detection requirements of MTX in different application scenarios,we modified Cy5 at the 5’ end of MTX specific aptamer HMX32,and gold nano-stars at the 3’ end of its complementary chain.At this point,the fluorescence of Cy5 was quenched,but the SERS signal was enhanced.Binding of MTX made HMX32 free from GNSs,thus fluorescence increased and SERS signal decreased.Based on this,we developed aptasensor HMX-SNA,which realized sensitive and specific detection of MTX.The dual-mode detection method improved the accuracy of the detection results,and meet the detection needs of MTX in different application scenarios.HMX-SNA is of great significance to further explore the design method of nucleic acid aptamer sensor,and the feasibility of GNSs and SERS signals for in vitro detection in clinical samples.4.Design of Aptamer-based signal amplification strategy for MTX detection in serumAs nucleic acids,aptamers have strong programmability and can be applied to various signal amplification technology.By combining branched DNA and CRISPR signal amplification with aptamer,dual signal amplification sensor HMX-c BCas was constructed to explore the possibility of MTX excretion delay detection.The results showed that the branched DNA structure significantly improved the detection sensitivity compared with no signal amplification and linear DNA.Moreover,the trans-cleavage activity of CRISPR-Cas12 a was greatly enhanced in the reducing environment.The successful design of HMX-c BCas lays a foundation for further development of MTX aptamer sensors with higher sensitivity.5.Design of primer exchange reaction based highly sensitive aptasensor for MTX detectionPER uses short DNA primers and catalytic DNA hairpin templates,with the help of strand displacement polymerase,to generate long tandem repeat single-stranded DNA of any specified sequence in a programmable,autonomous and stepwise manner,which has been used for signal amplification of immunofluorescence and fluorescence in situ hybridization.Combining aptamer and PER,an ultra-sensitive MTX detection sensor(Apt-PER)was constructed.Highly sensitive detection of MTX in 10% serum was achieved by rigorous optimization of reaction conditions with a linear detection range of 0.05 ～ 2 μM,and a detection limit of 0.0124 μM,corresponding to 0.124 μM in 100% serum.Compared with sensors HMX38-3,HMX-SNA,and HMX-c BCas,Apt-PER had higher sensitivity;compared with traditional detection technologies,Apt-PER had great advantages in detection cost,sample pretreatment process and other aspects.Above results indicated that Apt-PER is suitable for detecting the whole process of MTX excretion delay,and provides a new direction for the detection of MTX in clinical blood concentration monitoring. |
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