Aptamer-field-effect transistors overcome Debye length limitations for small-molecule sensing

Abstract

Detection of analytes by means of field-effect transistors bearing ligand-specific receptors is fundamentally limited by the shielding created by the electrical double layer (the "Debye length" limitation). We detected small molecules under physiological high-ionic strength conditions by modifying printed ultrathin metal-oxide field-effect transistor arrays with deoxyribonucleotide aptamers selected to bind their targets adaptively. Target-induced conformational changes of negatively charged aptamer phosphodiester backbones in close proximity to semiconductor channels gated conductance in physiological buffers, resulting in highly sensitive detection. Sensing of charged and electroneutral targets (serotonin, dopamine, glucose, and sphingosine-1-phosphate) was enabled by specifically isolated aptameric stem-loop receptors.

Fig. 1.. Isolation of stem-loop aptamer receptors.

(A) Schematic of field-effect transistor surface chemistry. (B) Layer-by-layer composition of FETs, FET microscope image, and photograph of experimental setup. (C) Oligonucleotide libraries (N_m, with random regions m from 30 to 36 nucleotides, flanked by constant regions and oligonucleotide primer regions for PCR amplification) were attached to agarose-streptavidin columns via biotinylated (B) complementary sequences. Exposure to targets (red sphere) causes elution of aptamers in which stems are stabilized. These sequences are preferentially amplified. Exposure to countertargets (alternate shapes) eliminates cross-reactive sequences. Aptamers for (D) dopamine (K_d=150 nM), (E) serotonin (K_d=30 nM), (F) glucose (K_d=10 mM), and (G) S1P (K_d=190 nM) were isolated. Solution-phase SELEX selected for aptamers that were directly converted to sensors. The complementary oligonucleotide was labeled with a quencher instead of biotin, while the aptamer was labeled with a fluorophore, leading to adaptive binding sensors with responses shown in (H to K). Fluorescence responses indicate selectivities of dopamine, serotonin, and glucose aptamers in the presence of specific vs. nonspecific targets. Fluorescence-concentration curves were the result of N=3 measurements with SEMs too small to be visualized in the graphs shown.

Fig. 2.. Electronic small-molecule detection using aptamer-functionalized field-effect transistor (FET) sensors

(A) Responses of FET sensors functionalized with the new dopamine aptamer (K_d=150 nM, full strength phosphate-buffered saline (1X PBS)) or its scrambled sequence, compared to FET responses with a previously reported dopamine aptamer (K_d=1 μM, 0.1X PBS) (2). (B) New dopamine aptamer and scrambled aptamer-FET responses to dopamine in artificial cerebrospinal fluid (1X aCSF). (C) For serotonin-aptamer-FETs, serotonin in 1X aCSF led to concentration-dependent responses, while scrambled serotonin sequences showed negligible responses. (D) New dopamine-aptamer-FET responses to 100 μM norepinephrine, serotonin, L-3,4-dihydroxyphenylalanine (L-DOPA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were negligible compared to dopamine (10 nM). (E) Serotonin-aptamer-FET responses to 100 μM dopamine, norepinephrine, 5-hydroxytryptophan (L-5-HTP), or 5-hydroxyindoleacetic acid (5-HIAA) were negligible compared to serotonin (10 nM). (F) Serotonin aptamer-FET sensitivities were shifted by altering ratios of amine-:methyl-terminated silanes for surface tethering. (G) Serotonin-aptamer-FETs after 1–4 h incubation in serotonin-free brain tissue followed by addition of serotonin exhibited reproducible responses with differentiable physiological concentrations. (H) Sphingosine-1-phosphate (S1P) aptamer-FETs showed concentration-dependent responses to S1P but not a phospholipid with similar epitopes or a scrambled sequence in 1x HEPES. (I) Glucose sensing in 1X Ringer’s buffer. Responses of glucose-aptamer-FETs were minimal or negligible for galactose, fructose, and a scrambled sequence. (J) Glucose aptamer-FET responses in mouse whole blood diluted in Ringer’s to construct a concentration curve. The red circle represents response in undiluted whole blood. (K) Glucose aptamer-FETs enabled differentiation of hyperglycemia in serotonin transporter deficient (KO) mice vs. wildtype (WT) mice by measuring glucose levels in diluted serum under basal and glucose challenged conditions. All calibrated responses were at gate voltage V_G = 100 mV. Error bars are +/− SEM with N = 6 (A–C, H, I) or N = 3 samples per group (D–G, J, K). *** P < 0.001 vs. countertargets; ** P < 0.01 KO vs. WT.

Fig. 3.. Aptamer-functionalized field-effect transistor (FET) mechanisms.

(A) Exposure of dopamine-aptamer-FETs to dopamine (artificial cerebrospinal fluid; 1x aCSF)) led to concentration-dependent reductions in source-drain currents. (B) For serotonin-aptamer-FETs, increasing concentrations of serotonin (1x aCSF) produced increases in source-drain currents. (C) Exposure of glucose aptamer-FETs to glucose (1x Ringer’s) led to reductions in source-drain currents. (D) The S1P aptamer-FET transfer curves (1x HEPES) increased in response to target concentrations. Transfer curves shown are representative of N=6 individual measurements. (E,F) Hypothesized mechanism of stem-loop aptamer target-induced reorientations in close proximity to semiconductor channels and within or near the Debye length. In (E), aptamers reorient closer to FETs to deplete channels electrostatically (e.g., dopamine, glucose). In (F), aptamer stem-loops reorient away from semiconductor channels increasing transconductance (e.g., serotonin, S1P). Schematics are idealized and do not reflect individual aptamer secondary structural motifs.

Fig. 4.. Changes in aptamer secondary structures upon adaptive binding to small-molecule targets.

(A) Circular dichroism spectroscopy of the dopamine aptamer upon target capture showed significant shifts indicating formation of a compact parallel G-quadruplex (1x aCSF). (B) By contrast, the serotonin aptamer showed a shift in peak positions indicating formation of an antiparallel G-quadruplex. Förster resonance energy transfer (FRET) between donor-, fluorescein (F), excited at 470 nm, and acceptor-, 5-carboxytetramethylrhodamine (T), labeled aptamers was monitored before and after target incubation. (C) For serotonin aptamers, donor fluorescence increased while acceptor emission decreased upon serotonin incubation suggesting that fluorophores move further away from each other upon target exposure. (D) Conversely, for glucose aptamers, the emission spectra for the acceptor increased while donor fluorescence decreased upon glucose exposure indicative of acceptor moving closer to donor enabling increased energy transfer. Stem-loop movement directions indicated by FRET for glucose vs. serotonin aptamers are consistent with their divergent FET transfer curve directions in Figure 3. (E) For glucose-aptamer-FETs with rigid double-stranded attachment stems (left), increasing distances from semiconductor surfaces by increasing the stem lengths (stem variants; right) resulted in (F) length-associated decreases in FET calibrated responses (1x Ringer’s solution). Spectra shown in A–D are representative of N=2 samples per condition; error bars in F are +/− SEMs with N=3 samples per group.