Liu et al. and electronics. This review focusses on one aspect of this collective effort: electrochemical biosensors. Among the various types of biosensors available, electrochemical biosensors are one of the most reliable, user-friendly, easy to manufacture, cost-effective and versatile technologies that can yield results within a short period of time, making it extremely NS-1643 promising for routine clinical testing. NS-1643 Ref.[94]SPGE/PPyNPs/AuNPs/Apt[Fe(CN)6]4?/3?Anti-IL-6 Apt6-mercaptohexanolSPGE+ Py+ LiClO4 polymerization: multipulse amperometrySPGE/PPyNPs+HAuCl4+ H2SO4 Au3+ reduction, NP formation: cyclic voltammetrySPGE/PPyNPs/AuNPs + Apt (thiolated): Au-S chemistry, multipulse amperometrySPGE/PPyNPs/AuNPs/Apt + MCH: Au-S chemistry, multipulse amperometry2.[105]Apt/AuNP/Au electrode[Fe(CN)6]4?/3?Anti-IL-6 AptHS-(CH2)11(OCH2CH2)3OHAu electrode/AuNP+ Aptamer(thiolated): Au-S chemistryEG3+ Au electrode/AuNP/Aptamer: Au-S chemistry3.[106]Au/Apt-MBMB (ox/red)Anti-TNF Apt6-mercapto-1-hexanol28-mer RNA Apt nucleotides are phosphorothiolated to protect from RNase5 NH2-Apt-(CH2)6SH 3 + MB-NHS 5 MB-Apt-(CH2)6SH 3 or Apt-MBApt-(CH2)6S-S-(CH2)6-OH 3 + TCEP+ H2O Apt-(CH2)6SH+ HS-(CH2)6-OH + TCEP = O5 MB-Apt-(CH2)6SH 3 + Au electrode: Au-S chemistry4.[107]Au/Apt-MBMB (ox/red)Anti-IFN Apt6-mercapto-1-hexanol3 NH2-(CH2)6-Apt+ MB-NHS 3 MB-NH-(CH2)6-Apt or MB-Apt5 OH-(CH2)6-S-S-(CH2)6-Apt+ TCEP 5 HS-(CH2)6-Apt+ OH-(CH2)6-SH+ TCEP = O5 SH-Apt-MB 3+ Au electrode: Au-S chemistry5.[108]Au/capture probeRecognition probe (Apt)H1-BtH2-BtSAv-ALP1-naphthol (red ox)Anti-IFN Apt6-mercapto-1-hexanolAu+ 5 SH-capture probe: immobilization with Au-S chemistryRecognition probe+ H1-Bt: hairpin opening, partial hybridizationPartially hybridized Bt-H1-recognition probe+ H2-Bt: hairpin opening, partial hybridization; resulting in a cascade of successive hybridization eventsBt-SAv: affinity bonding1-naphthyl phosphate (1-NPP) converted to 1-naphthol (1-NP) by ALP6.[109]Au/Apt[Fe(CN)6]4?/3?Anti-IFN Apt-mercaptoethanolDeactivation of RNase NS-1643 in solutions with diethyl pyrocarbonate (DEPC)Au electrode+ 5 SH-Apt (DNA/RNA)+ dithiothreitol+ pentanethiol/MgCl2/PBS: Apt immobilization, interspaced with pentanethiol7.[110]Au/Apt-MBMB(ox/red)Anti-TGF-1 Apt6-mercapto-1-hexanolMB-NHS+ 5 NH2-Apt: covalent linkage; MB-Apt formationAu+ 3 SH-Apt: Au-S chemistry5 NH2-Apt-SH 3 (DNA) has a phosphorothioated backbone on 5 adenine & cytosine nucleotides, to resist nuclease degradation Open in a separate window Common abbreviations: SPGE = Screen printed graphite electrode, PPyNP = Poly pyrrole nanoparticles, AuNP = Gold nanoparticles, H1, H2 = DNA hairpins, Bt= Biotin, SAv = Streptavidin, ALP = Alkaline Phosphatase, MB = methylene blue; Apt = Aptamer; MB-NHS = methylene blue, carboxylic acid, succinimidylester, EG3 = HS-(CH2)11(OCH2CH2)3OH, an oligo(ethylene oxide), TCEP = tris-(2-carboxyethyl)phosphine hydrochloride. Table 2 Aptasensors: Specifications and performance characteristics. Ref.[94]EIS0.33 pg/mL1 pg/mLC15 g/mLBSA, CEA, MUC1, MUC4, MUC16+IL-6/30 minIL-6 in PBSSpiked serum(6 assays)RSD = 3.42%–2. [105]EIS0.02 pg/mL0.02C20 pg/mLBSA-IL-6 in bufferSpiked artificial sweat(5 assays, at [IL-6] = 0.02 pg/mL) RSD = 14.1%After 2 weeks- 90% of initial signal retained for [IL-6] = 0.2 pg/mL90% of initial signal retained for [IL-6] = 0.02 pg/mL3. [106]SWV58 pM58 pMC6 nM-Sensor equilibration/30 min+TNF/15 minrTNF in spiked whole blood-Stable over 10 hAfter 6 cycles & regeneration/urea, 90% of original signal retained4. [107]SWV0.06 nM0.06C10 nMIgG, anti-IgG, BSA+IFN/15 minrIFN in HEPES bufferIFN in RPM1 culture mediaIFN in RPM1/serum–Regeneration/urea/1min + rinsing/diH2O; sensor can be reused more than 10 or more times5. [108]DPV0.3 nM0.5C300 nMPDGF-BB, BSA, IgG, CEA, IL-6IFN+ recognition probe/2 h+Au/capture probe/1 h+H1-Bt, H2-Bt/90 min, rinsing/10 min+SAv-ALP/30 min, rinsing/20 min+1NPP/3 minIFN in HEPES bufferIFN in RPM1 culture mediaIFN in RPM1/serum-After 2 weeks, no significant change in current responseRegeneration/NaOH/20 min + washing/diH2O/10 min; sensor can be reused more than 3 times6. [109]EIS500 fM (RNA Apt)1 pM (DNA Apt), 1.21 pM (DNA in FBS)-BSA, FBS+IFN/30 minIFN in PBSSpiked FBS—7. [110]SWV1 ng/mL1C200 ng/mLIL-2, IFN, BSA, IgG, TGF-2, TGF-3(real-time detection at constant flow)rTGF-1 in cell culture media DMEM— Open in a separate window Common abbreviations: PBS = Phosphate-buffered saline, BSA = Bovine Serum Albumin, CEA = Carcinoembryonic antigen, MUC = Mucin protein, Ig = Immunoglobulin protein, PDGF SAPKK3 = Platelet-derived growth factor, FBS = Fetal bovine serum, HEPES = (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), DMEM = Dulbeccos Modified Eagle Medium. Table 3 Enzyme-labeled immunosensors: Design and sensor preparation. Ref.[111]ITO/PDOP-AuNP/Ab1HRP-Ab2-AuNP-PDOP@CNTH2O2/H2OAnti-IL-6 Ab1, Ab2BSAITO + Dopamine + AuNP: polymerization of dopamine, AuNP absorptionITO/PDOP-AuNP + Ab1: physical absorptionCNT + dopamine + HAuCl4: Polymerization of dopamine, formation & absorption of AuNPHRP+ Ab2-AuNP-PDOP@CN: Physical absorption2. [95]PG/PDDA/GSH-AuNP/Ab1/BSABt-Ab2SAv-HRPHydroquinone/benzoquinoneH2O2/H2OAnti-IL-6 Ab1, Ab2 GSH + AuNP: Au-S chemistryGSH-AuNP + Ab1: amide bond formation with EDC/NHS catalystBt-Ab2+ SAv-HRP: affinity bonding3. [113]ITO/PDDA/AuNP/Ab1AuNP-Ab2-Bt-SAv-HRPHydroquinone/benzoquinoneH2O2/H2OAnti-IFN Ab1, Ab2BSAITO electrode + PDDA: drop-castingITO/PDDA(+ve) + AuNP(-ve): electrostatic adsorptionITO/PDDA/AuNP + Ab1: adsorptionCitrate reduction of HAuCl4: AuNP formationHRP-Ab2+ AuNP: adsorptionBt+ SAv: affinity bonding4. [114]SPCE-Phen-Ab1Bt-Ab2SAv-HRPHydroquinone/benzoquinoneH2O2/H2O Anti-IFN Ab1, Ab2BSAp-aminobenzoic acid (p-ABA) + NaNO2/HCl p-ABA diazonium NS-1643 saltSPCE + p-ABA diazonium salt: electrochemical reduction; grafting with CVSPCE-Phe-COOH + Ab1: amide bond formation with EDC/NHS catalyst5. [115]Mb-Ab1Bt-Ab2SAv-HRP-SPCEHydroquinone/benzoquinoneH2O2/H2OAnti-TGF-1 Ab1, Ab2EthanolamineMB-COOH+ Mix&Go polymer + Ab1: covalent immobilizationSAv+ Bt: affinity bondingMB-Ab1-TGF-1-Ab2-HRP separated with magnet at the base of SPCE6. [116]SPCE/SAv-Bt-Ab2V-Phen-SWCNT(-HRP)-Ab1(C5H4N(CH2)2NH2)1+/2+ or V1+/2+H2O2/H2OHydroquinone/benzoquinoneAnti-TGF-1 Ab1, Ab2BiotinSWCNT + p-aminobenzoic acid (p-ABA) NS-1643 + isoamylnitrile/NMP SWCNT-Phe-COOH; grafting4,4-bipyridine+ 2-bromoethylamine/CH3CN 1-(3-aminoethyl)-4,4-bipyridinium bromide ((C5H4N(CH2)2NH2)22+), a viologen V(C5H4N(CH2)2NH2)22+ + SWCNT-Phe-COOH .