Electronic hybridization detection in microarray format and DNA genotyping

We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.


I. CORRECTION OF RELATIVE DRIFT IN THE MEASUREMENT OF FIG.4
Significant drift occurred during the measurement presented in Fig.4 of the main text.
Comparing the transistors of the array that were covered by PLL but not by DNA probes, we observed that their threshold voltages shifted differently in time. We corrected this relative drift by subtracting a quadratic polynomial of the form y = ax 2 + bx + c, where x stands for the index of the FET in the array with x ∈ [1, 96]. The coefficients a = −1.39 × 10 −6 V, b = 1.23 × 10 −4 V and c = 1.37 × 10 −2 V are obtained from a fit to the PLL region of the array. Figures S1 and S2 display the voltage difference −∆U SE = U i 10 − U f 10 corresponding to an initial and a final electronic measurement in 10 µM KCl. The two panels of figure S1 correspond to sequential hybridizations on the same chip. In figure S1 (a), the average −∆U P 1,P 2 equals 6.8 mV. This positive value corresponds to specific hybridization of T1 targets. In figure S1 (b), the average −∆U P 1,P 2 is -2.5 mV. This negative value corresponds to specific hybridization of the target T2 oligomers. Figure S2 shows 35delG genotyping using 10 µM KCl in the electronic measurements. The average −∆U P 1,P 2 here equals 8.6 ± 2.0 mV.
Results of various hybridization measurements are summarised in Table S1. As for electronic measurement at 25 mM KCl, reproducible detection is achieved in the differential signal ∆U P 1,P 2 and not in the individual signals ∆U P 1 , ∆U P 2 , ∆U P 1,P LL and ∆U P 2,P LL .
For measurements with 25 mM electrolyte, the average of the electronic specificity signal 2 −∆U P 1,P 2 is 2.3 ± 0.7 mV, while in 10 µM this average amounts to 5.1 ± 1.6 mV. We can thus conclude that the signal amplitude is increased by the decrease in ionic strength. The signal to noise ratio however is not improved, because an increased variability is observed at 10 µM as compared to 25 mM. The increased variability of ∆U P 1,P 2 is tentatively attributed to the rinsing steps required when hybridization and detection are performed at different salt. Nevertheless, the majority (86 %) of FETs displayed the expected sign of the hybridization signal for detection at 10 µM. For the Tas-PCR product hybridization presented in Fig. S2 and main text Fig. 4 we observe that the amplitude of the electronic signal is enhanced by a factor of 6.5 for the 10 µM KCl solution as compared to the 25 mM case.
We observe that the sign of the average ∆U P 1,P 2 is always negative for hybridization with target T1 and positive or null for hybridization with T2. This sign is thus the oligomer hybridization experiments presented in Table 1 and a fluorophore on its 5'extremity. A forward primer that is composed of three parts : first a 20 base barcode sequence to be used for subsequent hybridization, followed by a carbon spacer (C18) for polymerase blocking and finally a sequence to prime target DNA.
The description of steps (a)-(h) of Fig.S3 is as follows.
• a) Target double stranded DNA (dsDNA). Solid lines correspond to the region that is to be amplified.
• b) Denaturation of target DNA.
• c) Annealing of forward and reverse primers.
• d) Elongation by polymerase from 5' to 3'. This step ends the first PCR cycle.
• e) Denaturation and annealing steps. From this step on, only strands preparing the synthesis of exponentially amplified products are presented. Thus, we consider the synthetized DNA ended at either a fluorophore or a single stranded DNA (ssDNA) barcode (green strand).
• f) Elongation and polymerase blocking. On the lower strand, elongation is blocked when the polymerase arrives at the carbon spacer leaving the last 20 bases single stranded (green strand). These 20 bases serve as a tag or barcode for subsequent recognition by hybridization. This step ends the second PCR cycle.
• g) Denaturation and annealing. We consider the shorter synthesized DNA terminated by either a fluorophore or a ssDNA tag (green strand).
• h) Elongation. At the end of elongation, products generated exhibit a barcode on one extremity and a fluorophore on the other extremity. This step ends the third PCR cycle. The presented species is exponentially amplified in the subsequent cycles.  (1) Denaturation (2) Annealing  Table S3. Sequences of the DNA oligonucleotides used in this work.

VII. QUANTITATIVE FLUORESCENCE ANALYSIS OF THE MICROARRAYS
To analyze the quality of our spotted microarrays, we investigated the hybridization signals by fluorescence using a commercial scanner (Innoscan 710, Innopsys, Carbonne, France). Using the same protocol for DNA immobilisation and blocking as for the FET arrays, two-dimensional microarrays were prepared on standard glass microscope slides. A typical fluorescence image is presented in Fig. S4. We spotted three columns of 10 spots, with base sequence P1 (left), P2 (middle) and P3 (right), respectively. Hybridization was performed for 15 min at room temperature with a solution containing 50 nM Cy5-modified-T1 and 50 nM Cy3-modified-T2 oligonucleotides in 25 mM KCl at pH7. The image is scanned with a resolution of 3 µm. Statistics of the fluoresence intensities is provided in Table S4.
Specific hybridization of target T1 on probe P1 and of T2 on P2 is seen, while non-specific interaction of T1 and T2 with the unrelated P3 sequence is comparatively low. Non-specific adsorption outside the spots is low as well. Overall, our arrays show performances that are typical of spotted fluorescence DNA microarrays. The spots are quite uniform in size, but still show some intra-spot and inter-spot variations in intensity. By reducing these variations it might be possible to further improve the electronic detection of hybridization.  Table S4. Statistical data of the fluorescence scan presented in Fig. S4. Line 4. Relative inter-spot variation of the specific signals. It is defined by for the left and middle column, respectively.
Line 5. Average ratio of specific signal to background. Background intensity corresponds to the average intensity around each spot.
Line 6. Average ratio of specific signal to non-specific signal. It is given by for the left and middle column, respectively.
Line 7. Average diameter of the specific spots. Figure S4. Image of a two color fluoresence scan of a typical microarray slide.
Red and green color corresponds to fluorescence signal measured with an excitation wavelength of 635 nm and 532 nm, respectively. We spotted three columns of 10 spots, with probes of base sequence P1 (left), P2 (middle) and P3 (right). Spacing of the spots is 200 µm.

VIII. SETUP AND PROCEDURES OF THE ELECTRONIC MEASUREMENTS
The setup and measurement procedures used in the electronic measurements has been described previously (main text reference 21) and is schematically represented in figure S5.
Drain currents I D of all transistors of an array are measured as a function of a source drain voltage U SD and a source electrolyte voltage U SE . The voltage U SE is applied between the common source and a Ag/AgCl reference electrode immersed in a KCl solution covering the array. We generate the two DC voltages U SE and U SD with a multifunction I/O board.
They are low-pass-filtered and applied to the FETs by an analog amplifier circuit and a multiplex-switch-unit controlled by an automated sequence programmed under Labwindows CVI (National Instruments).
Two electronic measurements are required to obtain a ∆U SE value. We used the protocol presented in figure S6 to determine the values of ∆U SE in 10 µM KCl solution and ∆U SE in 25 mM KCl solution that correspond to a DNA hybridization in 25 mM KCl.  Figure S5. Recording setup used for the differential detection of DNA. The FET array has one drain connection for each of its 96 transistors and one common source contact.