Electric field directed nucleic acid hybridization on microchips
Electric field directed nucleic acid hybridization on microchipsCarl F. Edman, Daniel E. Raymond, David J. Wu, Eugene Tu, Ronald G. Sosnowski, William F. Butler, Michael Nerenberg and Michael J. Heller*
Nanogen, 10398 Pacific Center Court, San Diego, CA 92121, USA
Received September 11, 1997;Revised and Accepted November 3, 1997
ABSTRACT
Selection and adjustment of proper physical parameters enables rapid DNA transport, site selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. However, only a subset of buffer species produce both rapid transport, site specific concentration and accelerated hybridization. These buffers include zwitterionic and low conductivity species such as: d- and l-histidine; 1- and 3-methylhistidines; carnosine; imidazole; pyridine; and collidine. In contrast, buffers such as glycine, [beta]-alanine and [gamma]-amino-butyric acid (GABA) produce rapid transport and site selective concentration but do not facilitate hybridization. Our results suggest that the ability of these buffers (histidine, etc.) to facilitate hybridization appears linked to their ability to provide electric field concentration of DNA; to buffer acidic conditions present at the anode; and in this process acquire a net positive charge which then shields or diminishes repulsion between the DNA strands, thus promoting hybridization.
Microchip-based nucleic acid arrays now permit the rapid analysis of genetic information by hybridization (1 -6 ). Many of these devices take advantage of sophisticated silicon manufacturing processes developed by the semiconductor industry over the last 40 years. In these devices, many parallel hybridizations may occur simultaneously on immobilized capture probes. Stringency and rate of hybridization is generally controlled by temperature and salt concentration of the solutions and washes. Such `passive' micro-array approaches have several limitations. First, as all nucleic acids are exposed to the same conditions simultaneously, capture probes must have similar melting temperatures to achieve similar levels of hybridization stringency. This places limitations on the length, GC content and secondary structure of the capture probes. Second, as the rate of hybridization is proportional to the initial concentration of the interrogated solution, high concentrations are required to achieve rapid hybridization. Third, because of difficulties controlling hybridization conditions, single base discrimination is generally restricted to oligomers of 20 bases or less with centrally placed differences (1 ).
In an attempt to circumvent these limitations, we have developed microchip-based hybridization arrays that utilize electric fields as an independent parameter to control DNA transport, enhance hybridization, and improve the stringency of nucleic acid interactions (7 ,8 ). We refer to these as `active' microelectronic devices in that they exploit both microfabrication and microelectronic technology, and that they directly affect the hybridization reactions. In addition to salt, pH, temperature and chaotropic agents, the electric field strength allows another precisely controlled and continuously variable parameter for adjustment of hybridization interactions.
Electronic addressing and/or hybridization is carried out by selective application of a DC positive bias to the individual microelectrodes beneath the selected test sites. This causes rapid transport and concentration of negatively charged nucleic acid molecules over selected locations on the microelectronic array. The nucleic acid (DNA, RNA, polynucleotides, oligonucleotides, etc.) may then be immobilized by direct attachment to the permeation layer overlying the micro-electrode or by hybridization to a previously addressed and attached nucleic acid. This rapid concentration leads to a dramatic reduction in time for hybridization when compared to passive hybridization techniques. Thus, hybridization occurs in seconds rather than hours. Reversal of the electric potential then allows rapid removal of unhybridized molecules, as well as continuous adjustment of stringency.
We have experimentally determined several factors that facilitate this process. A crucial role is played by the ion-permeable layer which overlies the micro-electrodes. This layer allows attachment of nucleic acids. Ultimately, this enables single-stranded nucleic acids to be `electronically targeted' above these micro-electrodes and hybridized to the anchored complementary oligonucleotides while being protected from the reactive environment present at the electrode surface. The structure of typical arrays and permeation layers has been described previously (7 ,8 ).
Another important consideration is the composition of the transport and hybridization buffers. To facilitate rapid movement of nucleic acids by free solution electrophoresis, we have utilized low conductivity buffers. To achieve low conductivity and preserve good buffering capacity, we have used zwitterions that have no net charge near neutral pH. These buffers typically possess conductivities less than 100 microSiemens per cm (µS/cm). Buffers commonly employed in molecular biology have conductivities a thousand-fold greater, e.g. 6× sodium chloride/sodium citrate (SSC) (9 ). Many of these low conductivity and zwitterionic buffers with no net charge do not optimally shield nucleic acid phosphodiester backbone charges and, therefore, under passive conditions do not aid in hybridization. However, they do probably help to prevent self-annealing of denatured nucleic acids in the transport process. We have now discovered that some buffer species (histidine, etc.) do facilitate both transport and the accelerated hybridization of DNA at the micro-array test sites. We present herein our findings concerning the characteristics of the low conductivity and zwitterionic buffers which facilitate these processes.
Buffers and chemicals utilized were purchased from Sigma (St. Louis, MO), Aldrich, (Milwaukee, WI), ICN Biochemicals (Aurora, OH), Boehringer-Mannheim (Indianapolis, IN) or Calbiochem (San Diego, CA). Oligonucleotides were synthesized as before (8 ) or were purchased from Oligo Therapeutics (Wilsonville, OR). Peptide nucleic acid (PNA) oligonucleotide analogues were synthesized by PerSeptive Biosystems. Conjugation of functional groups and oligonucleotide purification has been described previously (8 ).
These have been described previously (8 ). In brief, the 5580 series APEX chip used for these experiments consists of a 5 × 5 array of 80 µm circular microelectrodes with 200 µm microelectrodes at each corner of the array. Chips were mounted on a micromanipulator stage and the microelectrodes activated by a power supply and appropriately controlled relay switches. Fluorescent labeled oligonucleotides on the chip were visualized using oblique illumination with two 594 nm HeNe lasers and the images were quantified using either NIH Image or IPLab Spectrum software packages.
Two biotinylated capture oligonucleotides, ATA5 and ATA4 (8 ) were electronically transported and localized at adjacent microelectrodes. The solution was removed and the chip washed three or four times in test buffer. Buffers employed in these studies are listed in Table 1 and were utilized at the listed pH values and concentrations presented. The l-isomers of amino acids were used, unless otherwise noted. The chip was equilibrated in test buffer for 5-10 min. Then fresh buffer containing 10 nM BODIPY-Texas Red labeled RCA5 (btrRCA5), an oligonucleotide complementary to ATA5 but not ATA4 (8 ) was applied. The 200 µm microelectrodes were employed as cathodes and one microelectrode within the array used as the corresponding anode. Accumulation was assessed by sourcing a 500 nA constant current to individual microelectrodes for 30 s and monitoring the accumulation of fluorescence at the positively-biased (anode) site in the microelectrode array. Signal arising from non-fluorescent sources (i.e., detector thermal noise, etc.) was subtracted. Experiments examining the effect of different currents upon signal accumulation were conducted in a similar fashion except that the applied current varied from 100 nA to 1 µA. Initial rates were calculated for the first 5 s of signal accumulation, during which linear fluorescence accumulation was observed in all buffers. For the comparison between conductivities of various buffers, data is presented as fold increase over initial fluorescence to account for chip-to-chip variation as well as laser illumination variations in signal levels. For other experiments, data is presented as attomol product versus time, after converting the observed fluorescence intensity to moles of labeled oligonucleotide. (Absolute calibration of the fluorescence signal intensity to the surface density of fluorophores was done by measuring fluorescence from 10-20 µm thin film solutions containing known concentrations of fluorescent labeled oligonucleotides layered over an unpatterned platinum surface.) Control experiments indicate that fluorescence measurements were unaffected by buffers and conditions employed.
aValues obtained from Budavari, S. (1989) The Merck Index, 11th Ed., Merck & Co., Inc., Rahway, NJ, unless otherwise noted. bpH of buffer solution measured in water at room temperature.
cRemelli, M., Munerato, C. and Pulidori, F. (1994) J. Chem. Soc. Dalton Trans., 2049-2056. dnd, not determined.
Following accumulation at one microelectrode location, the buffer was removed and fresh buffer containing btrRCA5 applied. An adjacent microelectrode was then sourced in a similar fashion. Upon completion, the buffer was removed and the chip washed between five and seven times with test buffer without btrRCA5. The pair of previously targeted microelectrodes were then illuminated and the fluorescence present at each site quantified. Control experiments utilized a bodipy-Texas Red labeled oligonucleotide complementary to ATA4 to verify the integrity and concentration of the ATA4 capture oligonucleotide. Percent hybridization efficiency was calculated as follows: (complementary oligonucleotide signal - non-complementary signal)/(signal present immediately prior to the end of the applied current) × 100.
The conductivities of test buffer solutions utilized for transport and hybridization were measured at room temperature using an Accumet 1.0 cm-1 glass conductivity cell connected to an Accumet Model 50 pH/Ion/Conductivity meter (Fisher Scientific). Buffer pH values were obtained using an Accu-pHast variable temperature combination electrode and the same model meter.
Antimony electrodes were fabricated using methods described elsewhere (10 ) with the following minor modifications. Theta glass (World Precision Instruments, Sarasota, Florida) was used to obtain a double barrel type electrode, plus all capillaries were hand pulled with a final tip diameter estimated to be 75 µm. The potential of the antimony electrode was measured relative to a Ag/AgCl reference electrode using the pH meter employed above. Calibration of the antimony electrode yielded a slope of 57 mV/pH, a value in good agreement with other literature values (10 -13 ). While viewed through a microscope, the antimony electrode was brought down to the surface of an agarose coated chip using a motorized X-Y-Z micromanipulator until the electrode was observed to bend. Afterwards the electrode was raised to a height just above the observed deflection point. The distance from the antimony electrode to the chip surface was estimated to be 7 µm. The antimony electrode tip was then positioned over individual microelectrode wells at an oblique angle so as to minimize diffusion effects (10 ). The pH readings were obtained using either 200 or 500 nA constant current in either 50 mM KCl, 50 mM histidine, 50 mM imidazole or 50 mM [gamma]-amino-butyric acid (GABA).
ATA5 was electrically targeted to the first four columns of the microarray on streptavidin/agarose coated chips prepared as above, and ATA4 was targeted to the remaining column. For passive hybridization, either 5.0 or 0.5 nM btrRCA5 in 5× SSC (1× = 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) was applied for 1 min, then the chips were washed six times in buffer and imaged. For the 3 min timepoint, fresh buffer containing btrRCA5 was applied and incubated for an additional 2 min to achieve 3 min cumulative incubation time. This approach was repeated for the remaining time points. A similar experiment was performed using 5 nM btrRCA5 in 50 mM histidine. For electric field hybridization, either 5.0 or 0.5 nM btrRCA5 in 50 mM histidine was applied. Five sites, four with ATA5 previously attached and one with ATA4, were electronically targeted using 3.1 V total (~500 nA/site). Following application of the current for the desired time period, the chip was washed six times in histidine and imaged. Other data points were obtained in a similar fashion by targeting fresh solutions of btrRCA5 in histidine to previously untargeted sites for the indicated times. In both passive and electric field mediated hybridization, the background signal present at the ATA4 sites was subtracted from the signal present at ATA5 sites.
Thirty µM biotinylated oligodeoxyribonucleotides or PNA analogues were microdeposited upon individual sites of a streptavidin/agarose coated microarray in 10% glycerol, 25 mM NaCl, 25 mM sodium phosphate, pH 7.4, using an Eppendorf Micromanipulator 5171 and Transjector 5246. Following a 30 min incubation, the chip was rinsed with water. Sequences are as follows:
DNA1: biotin-d(CACCTGCTTTGATAGCTG)
PNA1: biotin-O-O-CACCTGCTTTGATAGCTG, O = ethylene glycol linker
DNA2: biotin-d(GATGAGCAGTTCTACGTGG)
PNA2: biotin-O-O-TGTACGTCACAACTA
Reporter DNA: btr-d(CAGCTATCAAAGCAGGTG), btr = BODIPY-Texas Red
DNA1 and PNA1 are complementary to reporter DNA whereas DNA2 and PNA2 serve as controls to evaluate non-specific hybridization. Electronic hybridization was performed with 5 µl of DNA reporter probe in 50 mM GABA or 50 mM histidine. The chip was electronically activated for 10 or 30 s at 200 nA or 30 s at 500 nA. Following each electronic hybridization, the chip was rinsed several times with the same buffer. After the final electronic hybridization, the chip was washed in a mixture of 0.2× STE (20 mM NaCl, 2 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, pH 8.0) and 0.1% sodium dodecyl sulfate, then 0.2× STE alone and a final image taken. Hybridization efficiency was calculated as above.
Biotinylated ATA5 and ATA4 were electronically targeted to adjacent sites upon streptavidin/agarose coated microarrays. Buffer consisting of 100 nM btrRCA5 in 50 mM histidine at its pI value (pH [approx] 7.5) or adjusted to either pH 6.0, 5.0, 4.0 or 3.0 with HCl, was added to each chip and allowed to react for 10 min. A parallel hybridization was done in 6× SSC. After this hybridization period, the chips were washed in 0.2× STE, 0.1% sodium dodecyl sulfate, as above and the remaining fluorescence at each site quantitated. Fluorescence values present at ATA4 sites were subtracted from adjacent ATA5 sites and the remaining fluorescence converted to attomol btrRCA5 specifically hybridized. Additional experiments repeated this methodology and also examined buffer adjusted with concentrated acetic acid (at the pI and at pH 6 and 5). Similar results were observed.
To promote hybridization of complementary nucleic acid in a dilute solution, the sample must be concentrated over the capture sequence. This causes a local increase in the Cot value, increasing the rate of hybridization by mass action. Under constant current conditions, charge is transported by all ionic species in the solution and thus buffer conductance determines the proportion of the current carried by the oligonucleotide. To measure this, fluorescent labeled reporter oligonucleotides were electronically targeted to locations where either complementary or non-complementary oligonucleotides had previously been attached. Accumulation of fluorescent signal over time in response to the applied current was then assessed for several different buffers (Table 1 ). We noted that accumulation eventually reaches a plateau. Therefore, to accurately measure the effects of conductivity, we have measured the initial rate of accumulation. Figure 1 displays analysis of a representative selection of buffers. The initial increase in fluorescent signal is compared to the inverse of buffer conductivity (i.e., solution resistance). This plot shows a roughly linear relationship between the solution resistance and the accumulation of signal. The more conductive the solution, the slower the rate of oligonucleotide accumulation.
The effect of electronic concentration of oligonucleotides on hybridization rate is shown in Figure 3 . In this experiment, an oligonucleotide was first electronically directed to and then anchored at selected sites of the microarray. The corresponding fluorescent labeled complementary oligonucleotide was introduced in either a high salt buffer (5× SSC), and allowed to passively hybridize (filled circles and squares), or was introduced in 50 mM histidine and electronically targeted to the capture oligonucleotide (open circles and squares). As shown, the electronically targeted oligonucleotide hybridization rate was 30- to 40-fold greater than the passive hybridization rate. No hybridization was observed using histidine in the absence of an applied electric field, irrespective of the time allowed (filled triangles). Thus, concentration alone may not fully explain the marked increase in efficiency of oligonucleotide hybridization in the electric field.
aData are presented as value ± SEM.
bFluorescent signal present at completion of electronic targeting.
cEfficiency = [(specific hybridization)/(transported)] × 100.
As noted above, those buffers that successfully supported hybridization have titratable substituents with pKa values which are at or near neutral pH. This could be particularly important since acid is generated by electrolysis over the anode (capture site) during transport. To evaluate the effect of pH on hybridization, a micro-pH electrode, approximately 75 µm in outside diameter, was positioned at a constant distance just above the surface of the permeation layer over individual electrode sites. Since the hydrolysis of water generates protons at the electrode (or within the electrochemical double layer), a proton gradient extends from the electrode surface through the permeation layer and out into the solution. Therefore, pH measurements shown in Figure 6 may be somewhat higher than those experienced by the oligonucleotides which are anchored in the topmost portion of the permeation layer. In the absence of buffer, a dramatic decrease in the pH was observed even at low current levels, 200 nA (Fig. 6 A). At 500 nA, the unbuffered system demonstrated an extremely rapid drop in pH (Fig. 6 B) and generated gas bubbles. In contrast, GABA was somewhat more effective in buffering the pH at low currents. Histidine and imidazole proved to be much more effective buffers maintaining the pH over the surface of the permeation layer above pH 5 for currents of either 200 or 500 nA.
The application of electric fields to microscale oligonucleotide arrays allows great acceleration and exquisite control over hybridization reactions. These advantages are augmented by the use of low conductivity or certain zwitterionic buffers. In general, these low-ionic strength buffers allow efficient transport of oligonucleotides to discrete sites in the presence of an applied current. These buffers do not support passive hybridization and, therefore, electronic control permits hybridization only at programmed sites. In addition to the mass action effect of concentration, our results suggest that the electrochemically-mediated production of positively charged buffer ions also facilitates the hybridization process. We have noted that a select subgroup of low conductivity buffers support hybridization under these conditions. Thus, we can separate transport from hybridization through the buffers and currents employed.
This programmed pH gradient allows discrete activation of hybridization zones and is a novel application for microelectronic devices. In our case, the buffering serves two roles: (i) it maintains the pH as close to neutrality as possible; and (ii) it actively participates in the reaction. In fact, if the pH is lowered below a critical threshold value, hybridization will be hindered. In short, in alleviating the detrimental pH effects created by hydrolysis at the anode, a species beneficial to the hybridization reaction is generated.
Interestingly, the efficiency by which these buffers support the hybridization process appears dependent upon the nature of the functional groups present. That is, once the criteria for possessing a buffering capacity within the hybridization window and the resultant generation of a positively charged species have been met, other functional groups may influence the hybridization process. The 5-fold increase in efficiency for histidine and related molecules over imidazole alone may reflect histidine's ability to sustain a positive charge on both the imidazole ring as well as the primary amine. Like simple dicationic salts such as Mg++, this double positive charge may be more efficient at diminishing backbone repulsion. Further modification of these zwitterions may lead to additional increases in hybridization efficiency.
The utility of these microelectronic devices is just beginning to be explored. As a group, they utilize lower quantities of reagents and often have significantly greater sensitivity and speed than conventional procedures. Ultimately, they may allow replacement of our present macro-scale molecular biology equipment by suitable microscopic devices.
The authors gratefully acknowledge the intellectual contributions and support of Drs Tina S. Nova and James P. O'Connell throughout the course of this work and for helpful insight into electrochemical processes by Eve Fabrizio.
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