Bioconjugation techniques for microfluidic biosensors

Abstract

We have evaluated five bioconjugation chemistries for immobilizing DNA onto silicon substrates for microfluidic biosensing applications. Conjugation by organosilanes is compared with linkage by carbonyldiimidazole (CDI) activation of silanol groups and utilization of dendrimers. Chemistries were compared in terms of immobilization and hybridization density, stability under microfluidic flow-induced shear stress, and stability after extended storage in aqueous solutions. Conjugation by dendrimer tether provided the greatest hybridization efficiency; however, conjugation by aminosilane treated with glutaraldehyde yielded the greatest immobilization and hybridization densities, as well as enhanced stability to both shear stress and extended storage in an aqueous environment. Direct linkage by CDI activation provided sufficient immobilization and hybridization density and represents a novel DNA bioconjugation strategy. Although these chemistries were evaluated for use in microfluidic biosensors, the results provide meaningful insight to a number of nanobiotechnology applications for which microfluidic devices require surface biofunctionalization, for example vascular prostheses and implanted devices.

Fig. 1

Schematic of bioconjugation chemistries

Fig. 2

FTIR spectra of silanetreated substrates (a) GOPS and (b) APTMS

Fig. 3

Immobilization density of capture probes before (dotted) and after (solid) microfluidic flow. Values are means of at least four determinations with error bars representing standard error

Fig. 4

Hybridization density of target probes. Values are means of at least four determinations with error bars representing standard error

Fig. 5

Hybridization efficiency. Values are means of at least four determinations with error bars representing standard error

Fig. 6

Stability of immobilized capture probes. a APTMS-GL and CDI functionalizations, b DEND-GL, GOPS, and APTMS-DEND functionalizations. Values are means of at least three determinations with error bars representing standard error