Abstract
Detection of genetic mutations leading to hematological malignancies is a key factor in the early diagnosis of acute myeloid leukemia (AML). FLT3-ITD mutations are an alarming gene defect found commonly in AML patients associated with high cases of leukemia and low survival rates. Available diagnostic assessments for FLT3-ITD are incapable of combining cost-effective detection platforms with high analytical performances. To circumvent this, we developed an efficient DNA biosensor for the recognition of AML caused by FLT3-ITD mutation utilizing electrochemical impedance characterization. The system was designed by adhering gold-sputtered zinc oxide (ZnO) nanorods onto interdigitated electrode (IDE) sensor chips. The sensing surface was biointerfaced with capture probes designed to hybridize with unmutated FLT3 sequences instead of the mutated FLT3-ITD gene, establishing a reverse manner of target detection. The developed biosensor demonstrated specific detection of mutated FLT3 genes, with high levels of sensitivity in response to analyte concentrations as low as 1 nM. The sensor also exhibited a stable functional life span of more than five weeks with good reproducibility and high discriminatory properties against FLT3 gene targets. Hence, the developed sensor is a promising tool for rapid and low-cost diagnostic applications relevant to the clinical prognosis of AML stemming from FLT3-ITD mutations.
Highlights
Acute myeloid leukemia (AML) is a widely studied case of hematological malignancy involving the uncontrolled proliferation of myeloid progenitors with impeded differentiation capabilities [1]
Since FMS-like tyrosine kinase 3 (FLT3)-ITD is reported to occur in the JM domain of membrane-bound receptor tyrosine kinase (RTK), both the amino acid sequence and the FLT3 genesequence were analyzed in tandem to isolate the sequence region of the gene which corresponds to the JM domain (Figure 2A)
This manner of “reverse detection” whereby the capture probe targets an unmutated FLT3 gene sequence enables the proposed sensor system to effectively differentiate between normal and FLT3-ITD relevant acute myeloid leukemia (AML) samples based on the inability of the probe to hybridize with the mutated target sequence as compared to the normal target sequence
Summary
Acute myeloid leukemia (AML) is a widely studied case of hematological malignancy involving the uncontrolled proliferation of myeloid progenitors with impeded differentiation capabilities [1]. Among the several different mutations associated with AML progressions, genetic alterations occurring in the FMS-like tyrosine kinase 3 (FLT3) gene is the most common known genetic marker diagnosed in both adult and pediatric AML patients [2,3]. Mutation in the FLT3 gene, encoding the membrane-bound receptor tyrosine kinase (RTK), disrupts such protein’s native function, leading to overexpressions, continuous clonal expansions, and damage towards the mechanisms of hematopoiesis. Mutation in the FLT3 gene, encoding the membrane-bound receptor2toyfr1o7-. Sine kinase (RTK), disrupts such protein’s native function, leading to overexpressions, continuous clonal expansions, and damage towards the mechanisms of hematopoiesis relreevleavnatntot tcoelcledllifdfeifrfeenrteinattiioatnioanndansdursvuivrvailv[a4l].[F4L].TF3LmT3umtatuiotantsioanres caaretecgaotreigzoedrizinetdoitnwtoo tswubostuybp-etys;pienst;eirnntearlntaalntdaenmdemdudpulipcalitciaotniosn(sIT(IDT)Di)ninthteheggeenneetthhaatteennccooddeess tthhee JJuuxxttaammeemmbbrraannee ((JJMM))ddoommaaiinnaannddppooiinnttmmuuttaattiioonnssiinntthheeggeenneetthhaatteennccooddeesstthheetytyrroossinineekkininaasseeddoommaaiinnss ((TTKKDD))..FFLLTT33--IITTDDisisoobbsseerrvveeddttooppoosssseessssaahhigighheerrffrreeqquueennccyyooffininccidideennccee((aapppprrooxxiimmaatteellyy 2255%%ooffrereppoortretdedAAMMLLcacsaesse)sa)nadndgrgeareteartecrlincliicnailcsailgsniigfinciafinccaencoemcopmarpedarwedithwFitLhTF3L-TTK3D-TK[5D]. AAlldy,dtihtieoncoa-lloyc,ctuhrerecnoc-eococfuFrLreTn3c-IeToDf wFLitTh3o-ItThDer wmiuthtoathioenrsmsuctahtiaosntshseumchuatasttihonesmouftnauticolnesopohf onsumclienop(NhPosMm1i)na(nNdPDMN1)AamndetDhNylAtramnseftehryalstera3nAs(fDerNaMseT33AA()DaNreMfuTr3tAhe) rarepfuortheder[r6e,7p]o,ritneddic[6a,t7iv],einodfitchaetivr eimofptohretiarnicmepaosretaxncecleleanstegxecenleletinct bgieonmeatirckberiosmfoarrekaerrlsyfaonrdearrellyapasneddriealganposesids ioafgAnoMsiLs ionf pAaMtieLnitns.patients. F[L5]T.3F-ILTTD3-cIoTnDstcitountsetsitfuatitehsfufali,tihnf-ufrl,amine-fdraumpleicdautiponliscaotfiovnasrioabf lveanruiacbleleotnidueclleeontgidtheslwenhgitchhs dwehliivcehr dpeoloivrefrupnoctoironfuanlictytiotonatlhiteyJMto tdhoemJMaind, oremnadienr,irnegndtheerisneglft-hinehsieblift-oinryhisbyistotermy soyfstehme mofemthbermaneempbrroatneeinpdroistoerindedriesdo.rdTehreedre. sTuhletinregscuhltaingecshnaengaetsivneelgyaatifvfeclyt tahfefeacctttihveataiocntivoaftthioenprooftethine, pleraodteining, tloeaaduitnog-pthooasupthoo-pryhloastpiohnoraynlda,titohneraenfodr,et,hseigrenfaolr-ein, dsiegpneanl-dinendtepcelnludleanrt pcreolluiflearratpioronli(fFeirgautiroen1)(F[4ig,5u]r.eA1d)d[i4t,i5o]n. aAlldy,dtihtieoncoa-lloyc,ctuhrerecnoc-eococfuFrLreTn3c-IeToDf wFLitTh3o-ItThDer wmiuthtoathioenrsmsuctahtiaosntshseumchuatasttihonesmouftnauticolnesopohf onsumclienop(NhPosMm1i)na(nNdPDMN1)AamndetDhNylAtramnseftehryalstera3nAs(fDerNaMseT33AA()DaNreMfuTr3tAhe) rarepfuortheder[r6e,7p]o,ritneddic[6a,t7iv],einodfitchaetivr eimofptohretiarnicmepaosretaxncecleleanstegxecenleletinct bgieonmeatirckberiosmfoarrekaerrlsyfaonrdearrellyapasneddriealganposesids ioafgAnoMsiLs ionf pAaMtieLnitns.patients.
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