Lateral Flow Aptasensor for Small Molecule Targets Exploiting Adsorption and Desorption Interactions on Gold Nanoparticles

A lateral flow assay (LFA) can provide a rapid and cost-effective means to detect targets in situ; however, existing LFA formats (predominantly sandwich assays) are not suitable for small molecule targets. We present a new LFA design that probes the dissociation of aptamers from the surface of gold nanoparticles upon recognition of small targets. The target-induced removal of aptamer molecules from the surface of the colored particles results in the particles being captured on a test line comprised of the protein bovine serum albumin immobilized on nitrocellulose. On the other hand, in the absence of target, aptamer coated particles are protected from capture on the test line and are instead captured at a control line comprised of the protein lysozyme. This protein is strongly positively charged under measurement conditions and therefore captures all gold nanoparticles regardless of the presence of aptamers. The effectiveness and operation mechanism of this simply fabricated sensor was demonstrated by using a previously reported 35-mer aptamer for a small molecule, 17β-estradiol. The sensor exhibited nanomolar level of detection, excellent selectivity against potential interfering molecules, and robust operation in natural river water samples. The simplicity and performance of the sensor platform renders it applicable to a wide range of other aptamers targeting small molecules, as we demonstrated with a novel bisphenol A aptamer. Additionally, we show that our LFA design is not confined to the specific proteins used as test and control lines, provided that their charge is appropriate to modulate the interaction with aptamer-coated or bare nanoparticles

Ultrasensitive Colorimetric Detection of 17β-Estradiol: The Effect of Shortening DNA Aptamer Sequences

We report a strategy enabling ultrasensitive colorimetric detection of 17β-estradiol (E2) in water and urine samples using DNA aptamer-coated gold nanoparticles (AuNPs). Starting from an established sensor format where aggregation is triggered when target-bound aptamers dissociate from AuNP surfaces, we demonstrated that step-change improvements are easily accessible through deletion of excess flanking nucleotides from aptamer sequences. After evaluating the lowest energy two-dimensional configuration of the previously isolated E2 binding 75-mer aptamer (<i>K</i>_D ∼25 nM), new 35-mer and 22-mer aptamers were generated with <i>K</i>_D’s of 14 and 11 nM by simply removing flanking nucleotides on either side of the inner core. The shorter aptamers were found to improve discrimination against other steroidal molecules and to improve colorimetric sensitivity for E2 detection by 25-fold compared with the 75-mer to 200 pM. In comparing the response of all sequences, we find that the excess flanking nucleotides suppress signal transduction by causing target-bound aptamers to remain adhered to AuNPs, which we confirm via surface sensitive electrochemical measurements. However, comparison between the 22-mer and 35-mer systems show that retaining a small number of excess bases is optimal. The performance advances we achieved by specifically considering the signal transduction mechanism ultimately resulted in facile detection of E2 in urine, as well as enabling environmental detection of E2 at levels approaching biological relevance