Aptamer/Nanoparticle‐Based Sensitive, Multiplexed Electronic Coding of Proteins and Small Biomolecules through a Backfilling Strategy

Abstract

The development of sensitive and selective bioassays capable of monitoring important biomolecules in a multiplexed fashion is a major goal in biosensing. [1][2][3][4][5] In many cases, measurement of a single biomarker is not sufficient to diagnose and follow the disease status. Rather, simultaneous measurement of a panel of biomarkers is necessary to reach this goal; this has led to increasing research on multiplexed analysis of biomolecules. Various multiplexed detection schemes for DNA or proteins based on labels with distinct readouts, such as optically [6][7][8][9][10][11] or electrochemically [12][13][14][15] encoded quantum dots, multisegment nanowires, [16][17][18][19] dye-embedded microspheres, [20][21][22][23][24] and biobarcode nanoparticles, [25,26] have thus been demonstrated. Although multiplexed analysis of DNA or proteins is well developed, simultaneous monitoring of analytes with significant difference in sizes, for instance, small biomolecules and proteins, has rarely been exploited and remains a challenge mainly due to the lack of double-probe sandwich assay formats for the low-molecular-weight analytes.Herein, we report, for the first time, a novel aptamer/ nanoparticle-based backfilling strategy for one-spot simultaneous detection of proteins and small biomolecules, employing lysozyme and adenosine triphosphate (ATP) as the model target analytes. Our approach relies on target-induced release of aptamers from the DNA duplexes on a sensing surface, followed by backfilling hybridization of the resulting single-stranded DNA molecules with aptamers conjugated to the electrochemically encoded nanoparticles. Subsequent unique electrochemical (EC) signatures of the acid-dissolved nanoparticles at distinct potential positions with well-resolved peaks thus reflect the identities and concentrations of lysozyme and ATP. Compared with previously reported multiplexed detection schemes, our new analysis route for proteins and small biomolecules offers three clear advantages. First, simultaneous determination of biomolecules with distinct molecular weights (lysozyme, 14.3 kDa versus ATP, 507.2 Da) is achieved for the first time with electrochemically encoded nanoparticle tags. Second, in contrast to other common "signal off" assay formats for lysozyme [27][28][29] and ATP based on target-induced displacement [30][31][32] or conformational changes of the corresponding aptamers, [33][34][35][36] our multiplexed method is a "signal on" configuration, which exhibits improved sensitivity.[35] Third, the analytical signal amplification by the nanoparticle labels composed of a large number of metal ions and the high sensitivity of the voltammetric stripping detection lead to lowconcentration determination of the target analytes. The combination of these advantages facilitates the detection of biomolecules with distinct sizes in a multiplexed and sensitive manner.Our multiplexed analysis approach for proteins and small molecules based on the aptamer/nanoparticle bioconjugates and the backfilling strategy, illustrate...

Electrochemical nanomaterial-based nucleic acid aptasensors

“…[90]; a sandwich assay was developed for thrombin detection taking advantage of the amplification ability of AuNPs carrying multiplex CdS NPs, as reported in Refs. [91,92].…”

Aptasensors Based on Stripping Voltammetry

“…Therefore, electrochemical aptasensors have been extensively applied in analysis of small biomolecules (e.g., adenosine and ATP [21,22], cocaine [23,24]), proteins [25][26][27], disease pathogens [28][29][30], and cancer cells [29,31,32]. Some metal ions, such as As(III) [33] or Pb(II) [34,35], can also be detected using suitable aptasensors obtained by the SELEX procedure.…”

“…The backfilling strategy belongs to an indirect analysis. Yuan and his group developed a multi-analyte aptasensor through a backfilling strategy for simultaneous detection of ATP and lysozyme using aptamer/nanoparticle bioconjugates as labels [21]. In this backfilling strategy, CdS QDs and PbS QDs are firstly tagged with different DNA sequences, which are complementary with an ATP aptamer and a lysozyme aptamer, respectively ( Figure 3).…”

“…Aptamers are alternatives to antibodies for bioassays. They can specifically bind to a variety of target molecules, such as proteins [36,39,67,68], drugs [62], small biomolecules [21,22], and cells [28,31,43,69].…”

High throughput immunosenor based on multi-label strategy and a novel array electrode

“…Although significant progress has been made in multianalyte assay using array chip, it is still a challenge to further raise throughput because of the number of detection spots is limited by the substrate area. More recently, researchers had developed the multiple-label strategy to realize the goal of one-spot multianalyte assay212223. For example, Wang et al proposed a gensensor for simultaneous detection of multiple DNA targets based on different metal nanoparticles lables (ZnS, PbS, CdS) which could yield sensitive signals at different stripping voltammetric potentials with dissolving metal nanoparticles in nitric acid24.…”