Micro- and nanodevices integrated with biomolecular probes

Abstract

Understanding how biomolecules, proteins and cells interact with their surroundings and other biological entities has become the fundamental design criterion for most biomedical micro- and nanodevices. Advances in biology, medicine, and nanofabrication technologies complement each other and allow us to engineer new tools based on biomolecules utilized as probes. Engineered micro/nanosystems and biomolecules in nature have remarkably robust compatibility in terms of function, size, and physical properties. This article presents the state of the art in micro- and nanoscale devices designed and fabricated with biomolecular probes as their vital constituents. General design and fabrication concepts are presented and three major platform technologies are highlighted: microcantilevers, micro/nanopillars, and microfluidics. Overview of each technology, typical fabrication details, and application areas are presented by emphasizing significant achievements, current challenges, and future opportunities.

Keywords: Biomolecules; Biosensing; Cell adhesion; Cell isolation; Micro/nanofabrication; Micro/nanopillars; Microcantilevers; Microfluidic channels; Point-of-care diagnosis; Surface functionalization.

Fig. 1

Schematic representation of a biosensor and essential components. Biomolecular probes include antibody, DNA, aptamer, and extracellular matrix (ECM) proteins. Using micro/nanobiosensors, nucleic acids, proteins, bacteria, and cells can be detected, their amount can be measured and their activity can be analyzed based on interactions occurring through biomolecular probes immobilized on the transducer surface. Transducers convert biological signals into measurable signals which are processed and analyzed.

Fig. 2

Four major categories of receptors on cell membrane playing role in cellular adhesion, and antibody, DNA, aptamer strands as biomolecular probes. Immunoglobulins (Igs, antibodies) are primarily associated with immune system, whereas selectins are involved in white blood cell homing. Immunoglobulins and selectins show high affinities with rapid binding rates. Cadherins and integrins are mainly involved in cell–cell and cell–matrix interactions, respectively. DNA and aptamer strands have been used as biomolecular probes with high specificity.

Fig. 3

Probing cellular weight on microcantilevers. (a) A polystyrene bead is placed on the control arm and a stem cell is placed on the sensing arm. Double resonance frequencies are quantified in a single measurement. In this example, the mass of the polystyrene bead was measured as 44.5 ng and the mass of the mouse stem cell was measured as 97.4 ng. For details see Chan et al. (2013, . (b) Illustration of a cell adhered and spread on a microscale cantilever for cell detection and/or cellular mass measurement. The weight of the cell is sufficient to deflect microcantilevers and change their resonant frequency. This approach exploits differences in resonance frequency shift of the microcantilever after cell attachment and growth, since system frequency is reversely correlated with mass.

Fig. 4

Production of a typical microcantilever by using photolithography and plasma etching. (a) Fabrication process of differential silicon nitride (SiNi) cantilevers. Only major steps are shown: (i–v) Cross-sectional view, (vi) top view. (b) Scanning electron micrographs of the die and differential nanomechanical biosensors. Inset shows a higher magnification image of an individual differential nanomechanical biosensor.

Fig. 5

Micropillar substrates can be modified to change topographical environment of cells. (a, b) SEM images of micropillar substrates with different array configurations and dimensions. Change in micropillar size, height, and spacing affects topographical cues for the cells, which has implications in cell spreading, alignment, migration, and differentiation. (c) Depiction of a single cell adhered on microscale pillar structures via biomolecules on cell membrane. When cells attach, spread, and migrate on a substrate they apply traction forces to the substrate, which in this case results in deflection of micropillars and, thus enables calculation of traction forces.

Fig. 6

Fabrication and surface modification of micropillar substrates. (a) Micropillar fabrication: (i) Pattern formation on photoresist material (SU-8) through photolithography using a photomask, (ii) development of photoresist via wet etching, (iii) PDMS casting, (iv) PDMS cure, (v) second PDMS casting, (vi) second PDMS cure, and (vii) final pillar substrate. (b) Schematic drawing of surface modification: (i) Incubation of protein on a PDMS block, (ii) protein adsorption on stamp, and (iii) and (iv) microcontact printing for protein transfer to micropillar top surfaces.

Fig. 7

Micro/nanopillar substrates are versatile tools that can be adapted to the study of various biophysical phenomena. Micro/nanopillar structures are utilized for force measurement by exploiting pillar deflection due to traction of cells adhered on top surfaces of the pillars. (a) Traction force measurement of cells in static conditions, where cells attach and spread on micropillar substrate. (b, c) Pillar substrates can also be modified to study effects of change in biophysical environment of cells. (b) On stiff micropillars, with greater pillar diameter, cells spread on the substrate, whereas displaying a rounded morphology on soft micropillars with smaller pillar diameter. (c) On anisotropically stiff micropillars cells grow in the stiff direction. (d) Nanowire embedded pillar structures can be used to exert forces on cells that are adhered to micropillar top surfaces via an external magnetic field. (e) Traction force measurement of cells under flow conditions can be performed using micropillars to study cell–cell and cell–substrate interactions in vessels.

Fig. 8

Structural designs utilized for microfluidic devices. (a–d) Various designs for flow pathway adapted for; measuring shear dependence in (a) Hele-Shaw channels, and increased surface area to facilitate cell–surface interaction in (b) parallel flow channels, (c) rectangular prism channels, and (d) spiral channels. Moreover, internal structures can be utilized to enhance cell–surface interaction using (e) micropost approach, or (f) herringbone mixing design. Figure sources, used with permission: a and b, Sin et al. (2005); d, Vickers et al. (2012); e, Nagrath et al. (2007); and f, Stott et al. (2010).

Fig. 9

Effects of binding proteins on antibody orientation. (a) Different immobilization methods: Antibody binding to (i) Protein G and (ii) NeutrAvidin, (iii) GMBS, and (iv) directly to plain glass surface. (b) Illustration of the AFM analyses: (i) low antibody density, (ii) high density randomly oriented antibodies, and (iii) high density uniformly oriented antibodies. (c) Surface roughness of different immobilization methods determined by AFM. (d) Surface morphology is modified with different functionalization approaches. Figure source, used with permission: Wang et al. (2012).