Fabrication and measurement set-up
The measurement set-up for molecular detection comprises an FET and a reaction chamber placed on an extended gate (EG) (Figs. 1b and d). We fabricated FETs with an active current channel in nanoscale by facilitating conventional semiconductor fabrication techniques (Fig. 1c and Supplementary Fig. S1). The thickness ratio of the top (14 nm) and bottom (750 nm) oxide layers determines its figure of merit17. The top gate electrode is electrically connected to an indium tin oxide (ITO) layer working as the EG electrode (see Supplementary information 1 for details), wherein the gate potential change modulates the conductance in the FET. This allows for reliable data acquisition by eliminating any possible contamination or damage to the active channel of the FET. Furthermore, all the responses are transduced by an identical FET device, which enables direct comparison of individual signals measured with different analytes at various concentrations. To maximize the response amplitude, the back-gate voltage (VBG) was adjusted to ensure the maximum slope in the top gate response curve (Supplementary Fig. S3).
Real-time monitoring of SLB coverage
SLB formation by spontaneous rupture of small unilamellar vesicles [SUV; 1 μg/mL in deionized water (DIW)] was examined using the FET (Fig. 2a). The SUV solution comprises 95% 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC) and 5% 1-oleoyl-2-[12-biotinyl (aminododecanoyl)]-sn-glycero-3-phosphoethanolamine (B-PE). Figure 2b shows the potential variation of the top gate electrode converted from the obtained current during the SLB formation (see Methods). The observed saturation after the fourth injection of the SUV is consistent with the calculation that 0.4 μg of lipids is sufficient to completely cover the SiO2 surface of the EG (see Supplementary information 5 for details). An epifluorescence microscopy was used for the cross-validation of the SLB formation (the inset of Fig. 2b).
Figure 2c shows drain current (ID) as a function of VBG with different SLB compositions, for direct comparison with compositional variations. The graph displays a signal resulting from the SLB containing 1 mol% of negatively charged Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE)18 and shows a positive threshold voltage shift compared to the neutral SLB (DOPC 100%). Note that our SLB-FET platform demonstrates sufficient molecular sensitivity to distinguish compositional differences within the SLB. For a fluorescence recovery after photobleaching (FRAP) test, we prepared a sample with the mol% ratio of 94:5:1 of DOPC:B-PE:TR-DHPE. The bleached region was recovered to 80% level of the initial fluorescent intensity after 10 min (Fig. 2d). With the lateral diffusion property of the lipids and the SLB with this compositional ratio, two effective binding sites of a single avidin is assumed to be occupied avoiding steric hindrance19. The diffusion rate was calculated to be 1.43 ± 0.4 μm2/s with high uniformity over the SiO2-coated EG20, 21.
Measurements of biotin-avidin bindings and analytical models
To evaluate our SLB-FET as a biosensor operating in an ionic solution, we conducted time-lapse measurements during biotin–avidin binding. The SLB containing 5% of B-PE with 95% of DOPC was prepared on the EG surface19. After SLB deposition through SUV rupture in DIW, the outer buffer (OB) solution above the SLB was exchanged with 1× phosphate buffer saline (PBS) to mimic physiologic conditions. Note that the ion impermeability of the SLB preserves the ionic imbalance across the lipid membrane (Supplementary Figs. S4 and S5). Figure 3a shows a typical real-time trace obtained from sequential steps including SLB formation (markers 1 and 2), OB exchange from DIW into 1× PBS (marker 3), 800 pM-avidin injections (markers 4, 6, and 8), and 800 pM-Cholera toxin subunit B (CTxB) injections (markers 5 and 7). CTxB was applied as a negative control analyte. While CTxB injections showed no response, avidin injections led to clear top gate voltage change (ΔVTG ~ 4.7 mV in the first injection) (Fig. 3b).
The real-time responses of biotin-avidin bindings at different analyte concentrations ranging from 100 pM to 100 nM are shown in Fig. 3c and summarized in Fig. 3d. To account for the obtained responses, an analytical model was suggested by combining the Langmuir isotherm model with the information of capacitively induced charges on the gate electrode of the FET. The induced voltage change ΔVTG can be described as
![](data:image/png;base64,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)
where qA is the effective charge induced by molecular adsorption, [B]max is the number of binding sites on the lipid membrane, [A] represents the analyte concentration, and Keq is the equilibrium constant22. The Keq of 621.9 pM for biotin–avidin binding was determined from the fitting using Eq. (1) (solid line in orange, Fig. 3d), which is consistent with the literature19, 23. To determine binding parameters, a resistor-capacitor (RC) circuit model was combined with the first order Langmuir adsorption equation (see Supplementary information 7 for details). The time-dependent ΔVTG is then expressed as
![](data:image/png;base64,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)
where k1 and k-1 are the association and dissociation rate constants, respectively, Vp is the difference between the maximum sensor response (qA[B]max/CTG) and the maximum value of ΔVTG(t), and τ1 and τ2 are the RC time constants. The rate constants were calculated to be k1 = 1.64 ± 0.06 × 107 M-1s-1 and k-1 = 1.02 × 10-2 s-1 based on fitting with the obtained value of Keq (=k-1/k1) above (Fig. 3c). The response curve from the EG functionalized by biotin at an identical concentration without the SLB (circle in grey, Fig. 3d) shows a linear increase in the high concentration regime, which is attributable to the nonspecific binding24. In contrast, this nonspecific binding behavior was strongly suppressed by the SLB (circle in orange, Fig. 3d).
Figure 3e presents the sensor responses using different conditions for the buffer solution around the SLB. We differentiated the ionic strength of the OB with 0.01×, 0.1×, and 1× PBS, while keeping the DIW isolated underneath the SLB. Unlike the usual presumption that the sensing capability highly depends on the ionic strength of the solution, the responses to 800 pM avidin in different ionic buffer conditions were similar and within the error range of our measurements. The control experiment with 1× PBS buffer solution underneath the SLB inevitably demonstrates the importance of the ionic condition of the inner buffer (IB) in our SLB-FET sensing mechanism. In addition, [B]max and CTG are calculated to be 1.74×108 and 0.37 pF respectively in 5 mol% of the B-PE. With this, qA is obtained to be 2.59 × 10-23 C (≈1.61×10-4 electrons) from the fitting. This suggests a significant discrepancy with the previous result of about 3.43 electrons for a single avidin molecule in 1× PBS solution25. These counter-intuitive results exhibiting ionic strength independent FET signals led us to the following hypothesis: there could be a possible conformational change upon molecular binding, redistributing the electron density within the SLB (Fig. 3f).
Analysis on the electron density change in the SLB upon molecular bindings
To investigate the underlying sensing mechanism of our SLB-FET upon molecular binding, synchrotron X-ray reflectometry (XRR) was applied to acquire electron density profiles across the SLB (DOPC/B–PE = 95/5 in DIW) before/after avidin bindings26. The obtained electron density profiles from the reflectivity fits are presented in Fig. 4a (see Supplementary Fig. S9b for details). Despite the structural similarity, conformational changes to the free-standing SLB due to avidin binding are distinguishable, and are accompanied by the redistribution of electrons within the SLB. For quantitative analysis, differences in the electric field (∆E) and chemical potential (∆ψ) were calculated using Poisson’s equation (see Supplementary information 9 for details) (Figs. 4b, c, and d). The induced chemical potential difference, ∆ψ, via the avidin binding was 697 mV at the surface of the SiO2 layer. By considering a series of potential drops along the circuit (Fig. 4e), the potential change at the top gate was calculated as 12.2 mV. This is consistent with the experimentally measured value, qA[B]max/CTG of 9.38 ± 3.08 mV in the FET, considering the fact that the XRR experiment was conducted under DIW conditions (EDLDIW >> EDL1× PBS). Note that the ∆VTG caused by the charges above the SLB (>55 Å in Fig. 4b) is within the range of standard deviation in the FET-measurements. To further cement our understanding, we compared the signal responses of avidin variants (neutrAvidin: pI. 6.3, streptavidin: pI. 5–6, and avidin: pI. 10.5) with different isoelectric points (pIs) under 1× PBS at the identical concentration of 800 pM (Fig. 4f). Coterminous FET responses regardless of different pIs support our finding that the majority of effective signals originate from dipole field change (i.e., electron redistribution) within the SLB, not from the conjugated proteins themselves27. Note that this sensing mechanism in which the SLB works as a transducer allows the detection of the electroneutral analyte.
Table 1. Comparison of FET sensors for molecule detection
SLB?
|
Electron channel
|
Analyte
|
LOD*
|
Solvent type & conc.
|
Refs.
|
○
|
Si
|
Av*, S.Av*, N.Av*
|
100 pM
|
1× (100 mM) PBS
|
our work
|
○
|
CNT
|
S.Av
|
2.5 uM
|
10 mM PBS
|
(28)
|
○
|
CNT
|
S.Av
|
5 uM
|
1 μM PBS
|
(29)
|
○
|
OSC*
|
S.Av
|
10 nM
|
10 mM PBS
|
(30)
|
○
|
Graphene
|
Magainin 2
|
100 pM
|
10 mM NaF
|
(27)
|
○
|
Graphene
|
CTxB
|
12.5 nM
|
10 mM HEPES
|
(31)
|
×
|
Si
|
PSA*
|
75 fg/mL
|
1 mM PBS, 2 mM KCl
|
(32)
|
×
|
Si
|
Thrombin
|
330 pM
|
Acetate buffer
|
(33)
|
×
|
Si
|
PSA
|
150 fM
|
100 μM PBS, 100 μM KCl
|
(34)
|
×
|
Si
|
cTnT*
|
1 fg/mL
|
100 μM PBS
|
(35)
|
×
|
Si
|
PSA
|
1 pg/mL
|
1 μM PBS, 2 μM KCl
|
(36)
|
*LOD: limit of detection, Av: Avidin, S.Av: Streptavidin, N.Av: NeutrAvidin, OSC: Organic semiconductor, PSA: Phostate Specific Antigen, cTnT: Cardiac troponin T