Towards in vitro molecular diagnostics using nanostructures

Abstract

Nanostructures appear to be promising for a number of applications in molecular diagnostics, mainly due to the increased surface-to-volume ratio they can offer, the very low limit of detection achievable, and the possibility to fabricate point-of-care diagnostic devices. In this paper, we review examples of the use of nanostructures as diagnostic tools that bring in marked improvements over prevalent classical assays. The focus is laid on the various sensing paradigms that possess the potential or have demonstrated the capability to replace or augment current analytical strategies. We start with a brief introduction of the various types of nanostructures and their physical properties that determine the transduction principle. This is followed by a concise collection of various functionalization protocols used to immobilize biomolecules on the nanostructure surface. The sensing paradigms are discussed in two contexts: the nanostructure acting as a label for detection, or the nanostructure acting as a support upon which the molecular recognition events take place. In order to be successful in the field of molecular diagnostics, it is important that the nanoanalytical tools be evaluated in the appropriate biological environment. The final section of the review compiles such examples, where the nanostructure-based diagnostic tools have been tested on realistic samples such as serum, demonstrating their analytical power even in the presence of complex matrix effects. The ability of nanodiagnostic tools to detect ultralow concentrations of one or more analytes coupled with portability and the use of low sample volumes is expected to have a broad impact in the field of molecular diagnostics.

Fig. 1

An overview of various nanostructures (NSs) that are used in diagnostic assays. NSs with a blue background are used as supports to immobilize receptors or labels, while those with a yellow background function as labels. NSs in a green background function both as labels and as supports. The insets in white background show the major bioconjugation protocol used to immobilize receptors on the corresponding nanomaterial. The green antibody in the insets represents a generic biomolecule

Fig. 2

Mind-map of the various nanostructure-based biomolecule detection paradigms

Fig. 3

Schematics of labeled biomolecule detection. NP and CNT refer to 0D and 1D nanostructure respectively. a A classical approach involving a label (such as an organic dye) directly attached to the analyte. Nanostructure-based approaches: b a carbon nanotube (CNT) or c a nanoparticle acting as a label; d a nanoparticle loaded with dye molecules acting as a label

Fig. 4

Schematics of major label-free sandwich assays. a A classical approach––enzyme-linked immunosorbent assay (ELISA) showing the analyte sandwiched between an immobilized primary receptor and a labeled secondary receptor. Nanostructure-based approaches: b CNTs or NPs loaded with reporters (enzymes or dye molecules) act as labels; c the NPs attached to secondary receptors act as labels themselves. After electroless deposition of silver, they are detected by colorimetry (Ag) or by measuring the resistance (R) between two electrodes; d increasing the density of primary receptors by immobilizing them on nanoparticles or nanostructured surfaces; e Biobarcode assay: every analyte molecule is translated into numerous (n) oligonucleotides; f Nanobarcode assay: every analyte molecule is translated into a metal nanowire carrying a specific code. In both barcode assays, a unique code is deployed (Code A or Code B) for the corresponding analyte (A or B)

Fig. 5

Schematics of label-free direct assays (unique for nanostructures). In general, the receptors are immobilized on the nanostructures. The binding of the analyte induces changes in some physical property of the nanostructure that is sensitively measured. a Local surface plasmon resonance (LSPR) on nanoparticles or nanopyramids and b surface plasmon resonance (SPR) on metal films detected by measuring the absorption spectrum or the angle of the reflected light. c Molecular beacons, where nanoparticles quench the fluorescence of an acceptor dye. d Changes in resistance (R), impedance (Z) or current (I) of 1D nanostructures (carbon nanotubes or silicon nanowires) measured electrically