| Diarrhea shellfish toxins(DSTs)are the most widely distributed marine toxins in the world at this stage,posing a huge threat to human health and affecting economic development.Humans can cause a series of digestive symptoms including nausea,vomiting,and diarrhea after drinking water or eating seafood contaminated by DSTs.There are three main types of diarrheal shellfish toxins:Okadaic acid(OA),Dinophysistoxins-1(DTX-1),and Dinophysistoxins-2(DTX-2).Among them,DTX-1 is the most toxic and can not only destroy the integrity of intestinal epithelial cells,but also play an important role in the process of apoptosis.For long-term performance,DTX-1 can also promote the occurrence of some cancers.The toxicity of DTX-1 is mainly due to the inhibitory effects of serine/threonine-specific protein phosphatases(PPs)1 and 2A(PP1and PP2A).At present,many methods for detecting DTX-1 have appeared:bioanalytical method represented by MBA,physicochemical analysis method represented by HPLC,and enzyme-linked immunosorbent assay method.However,these existing detection methods have some irreparable defects,which is difficult to meet the actual needs at this stage.Recently,Dinophysis events have emerged in traditionally“DSP-toxin free”areas,which indicates that the detection of DSTs is increasingly urgent,and it is important to develop a new detection method that can overcome the shortcomings of traditional methods.Aptamer is a new type of molecular recognition element,whose binding pattern to targets is similar to antibodies.That is why be called"chemical antibody".Researchers combined it with various biosensor platforms to develop a variety of new detection methods that are widely used in various fields such as toxin detection,food safety,and diagnosis.We obtained DTX-1 aptamer N59,which can specifically bind to DTX-1 via magnetic beads(MB-SELEX)with the highest affinity(K_D=170 n M).Based on this,referring to the prediction results of the mfold web server for the aptamer N59 secondary structure,we truncated the aptamer N59 and obtained its core sequence N59a.The affinity between aptamer N59a and DTX-1 was significantly improved(K_D=64 n M)after optimization.In order to further explore the binding mode of aptamer N59a and DTX-1,we performed a series of experiments such as base truncation analysis,circular dichroism,and molecular dynamics simulation.The results show that the aptamer N59a formed the i-Motif structure spontaneously and bound to DTX-1 specifically.Biolayer interferometry(BLI)technology is a label-free technology that can provide real-time information on high-throughput biomolecular interactions which is widely applicated in many fields now.Therefore,this time we successfully used aptamer N59a as the core molecule to construct a DTX-1 aptamer sensor with high sensitivity and strong specificity within the BLI technology platform.The BLI biosensor can detect DTX-1 in 15minutes.It has a linear detection range of 60-200 n M and a limit of detection(LOD)of 614p M.Although there is a little cross-reaction with OA,it does not cross-reaction with other types of toxins such as GTX,STX,BTX and PTX.In addition,the BLI biosensor has good stability.We applied this BLI biosensor to the detection of DTX-1 in seawater,and the results showed that the recovery rate of this BLI biosensor was high(100.5%?106.9%)while the CV value was also can be acceptable(1.2%?5.2%).All in all,we have obtained a high affinity and strong specificity DTX-1 aptamer N59a by MB-SELEX.Based on this aptamer,we constructed a BLI aptamer biosensor,which has high sensitivity and strong specificity.Consequently,we believe that this new BLI biosensor is expected to provide a basis for updating the traditional DTX-1 detection method,and after further improvement,it will be applied to the rapid detection of DTX-1in actual marine environment. |
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