KR100591626B1 - Methods and Materials For Optimization Of Electronic Hybridization Reactions - Google Patents

Abstract

The present invention relates to findings relating to various parameters, electrolytes (buffers), and other conditions that enhance or optimize DNA transport rates, efficiency of DNA hybridization reactions, and overall hybridization specificity in microelectronic chips and devices. In particular, the present invention relates to a low-conductivity zwitterion buffer, in particular a buffer containing histidine amino acids prepared so that the concentration is about 50 mM and the pH is near pI or pI (pH of the isoelectric point = pH 7.47) by electrophoresis. It is about the discovery that it provides the optimum conditions for making the hybridization reaction efficient while speeding up the process. At least 10-fold hybridization efficiencies are obtained compared to the second best known buffer, cysteine. Test data demonstrated that the hybridization efficiency increased approximately 50000 times compared to cysteine.

Histidine buffer, electrophoresis, DNA transport, hybridization

Description

Methods and Materials For Optimization Of Electronic Hybridization Reactions

The present invention relates to buffers and electrolytes for use in electronic devices suitable for medical diagnostic, biological and other applications. More specifically, the present invention relates to buffers and electrolytes that are advantageously used for DNA hybridization assays performed in microelectronic devices for medical diagnostics.

Recently, there has been a growing interest in devices that use both microelectronic and molecular biology technologies. One such system is "ACTIVE PROGRAMMABLE ELECTRONIC DEVICES FOR MOLECULAR BIOLOGICAL ANALYSIS AND" of US patent application Ser. No. 08 / 146,504 (currently US Pat. No. 5,605,662), filed November 1, 1993, which is incorporated herein by reference. "DIAGNOSTICS". The system disclosed in this document will be referred to as APEX system. The APEX system can perform a wide range of functions that are beneficial for molecular biological responses such as nucleic acid hybridization, antibody / antigen reactions, clinical diagnostics, and biopolymer synthesis.

APEX-type devices use buffers and electrolytes for operation. Buffers are defined as chemical solutions that do not change well with the addition of acids or alkalis. See, eg, Dictionary of Biotechnology, 2nd edition, James Coombs, Stockton Press. As described in this document, buffers based on " traditional mineral salts (phosphates, carbonates) and organic acid salts (acetates, citrates, succinates, glycine, maleates, barbiturates, etc.) have been used in biological experiments. ".

It is an object of the present invention to find buffers and electrolytes which are advantageously used in molecular biological electronic devices for carrying out hybridization, reaction, diagnosis or synthesis.

Summary of the Invention

The present invention relates to various parameters, electrolytes (buffers), and other conditions that enhance or optimize the DNA transport rate, efficiency of DNA hybridization reactions, and overall hybridization specificity in our APEX microelectronic chips and devices. It's about discovery. In particular, the present invention comprises a low conductivity zwitterion buffer, particularly histidine amino acids prepared such that the concentration is between 10 and 100 mM, preferably about 50 mM and the pH is near pi or pi (pH of the isoelectric point = about 7.47). The discovery is that buffers provide optimum conditions for rapid DNA transport and efficient hybridization reactions. Hybridization efficiency in the present invention was at least 10 times higher compared to the second best known Cysteine buffer. Test data demonstrate that the hybridization efficiency increased about 50000 times compared to cysteine.
The conductivity of the buffer used in the present invention is preferably substantially less than 1 mS, more preferably substantially less than 500 μS, more preferably substantially less than 250 μS, even more preferably substantially less than 100 μS, It is preferable that the original pI of is substantially 7.4 or more.

1 is a plan view of a checkerboard device using histidine buffer.

The transport of DNA and other charged analytes by electrophoresis in various types of electrolytes / buffers involves various physical parameters. The particular device, for example the APEX device of the inventors described in US Pat. No. 5,605,662, cited above, is basically a DC (direct current) electric device that generates an electric field at the surface of the device. This electric field, in turn, results in the transport of charged molecules by electrophoresis between the microscopically biased (+/-) locations on the device surface. In contrast, so-called xenosensors (see eg "Optical and Electrical Methods and Apparatus for Molecular Detection" of WO 93/22678, eg, Genosensor, impedance sensors, Hollis et al.) And dielectrophoresis The apparatus (see, for example, Wasashizu, 25 Journal of Electrostatics, 109-123, 1990) uses an AC electric field. An important feature associated with the devices is that upon application of the AC electric field there is essentially no net current flow in any of the systems (ie no driving force by electrophoresis for the transport of charged molecules). In APEX-type devices, a significant net direct current (DC) flow occurs when voltage is applied, which is recognized as a "sign of electrophoresis." In electrophoresis, ions or charged particles move by electrical energy along the direction of the electric field gradient, and the relationship between current and voltage is important for this technique. The electrophoretic shift appears as the current conduction of the solution, which occurs macroscopically under the influence of the applied voltage and Ohm's law

V = RxI

Where V is the potential, R is the electrical resistance of the electrolyte and [VxA ^-1 = R (Ω)] and I is the current [A].

The electrical resistance of a solution is the inverse of the conductivity that can be measured with an electrical conductivity meter. Conductivity mainly depends on the type and concentration of ions in the buffer / electrolyte, so these parameters are of great importance for molecular biology techniques involving electric fields. Even though the generated electric field exists in the actual microenvironmental state, the basic current / voltage relationship is essentially the same for other electrophoretic systems and APEX technology.

APEX systems have unique features in terms of the various ways of supplying current and voltage, and how current and voltage scenarios have been found to improve the performance of the system. In particular, various DC pulsing methods (linear gradient and log unit gradient) are believed to improve the stringency of hybridization.

Electrophoretic Transport vs Ionic Strength

It is well known in the field of electrophoresis that the mobility of charged analytes (proteins, DNA, etc.) decreases in logarithmic units in inverse proportion to the square root of the ionic strength of the electrolyte solution (R. Kuhn and S. Hoffstetter). See Capillary Electrophoresis: Principles and Practice, Springer-Verlag, 1993, Figure 3.16 on page 83). For a given constant electric field, the analyte will be transported at a faster rate if the electrolyte concentration is reduced compared to the concentration of the analyte (protein, DNA, etc.). Similar results demonstrating the effect on single-stranded amino acids are described in JJ Issaq et al., Chromatographia, Vol. 32, # 3/4, August 1991, pages 155-161 (especially see FIG. ]. Results demonstrating the effect on DNA in various electrolyte solutions are described in PD Ross and RL Scruggs, Biopolymers, Vol. 2, pages 231-236, 1964 (especially FIG. 1 on page 232). )].

Ionic Strength / Conductivity Relationship

The ionic strength in the free, buffer action containing an electrolyte (sodium chloride, potassium chloride, etc.) completely dissociated anion and cation species in solution (such as ^{Na + <---> Cl - -} , K + <---> Cl) And conductivity are the same (ie conductivity will usually be proportional to ionic strength). In buffered electrolytes (phosphate, acetate, citrate, succinate, etc.) in the dissociated state (e.g. 2Na ⁺ <---> PO ₄ ^-2 ), the ionic strength and the normal conductivity will be the same (ie conductivity Is proportional to ionic strength). For buffered electrolytes (“excellent buffers” (MOPS, HEPES, TAPS, tricine, bisine), amino acid buffers, amphoteric electrolytes, etc.) that may include zwitterionic species (no net charge at pi), conductivity Will decrease about 10-fold per pH unit difference between isoelectric point (pI) and pKa. For example zwitterionic ^{state-conductivity} of the amino acid of (OOC-CH (R) -NH 3 +) is completely net positive charge (HOOC-CH (R "amino acid residue") -NH ₂ ⁺ <---> It would be about 1000 times lower than if it had X ⁻ ) or completely negative charge (Y ⁺ <---> ^- OOC-CH (R) -NH ₂ ). Thus, as the amino acid residues move away from pi, formal negative or positive charges will appear on the amino acids and the conductivity and ionic strength will begin to correlate. However, near pI or pI, the conductivity will be much lower than expected at a given ionic strength or concentration. Electrophoretic literature mentions that "excellent buffer" and amino acid buffer have "low conductivity at high ionic strength or high ion concentration" when used in the vicinity of pI or pI (Capillary Electrophoresis: Principles and Practice, page 88, Springer-Verlag, 1993). The actual conductivity of the commonly used electrophoretic buffer "Tris-borate" is significantly lower than expected from its ionic strength or ion concentration. This may be due to the "tris cations" and "borate anions" forming a relatively stable zwitterion complex in solution. The conductivity of the 100 mM tris-borate solution was measured at 694 μS / cm, which is about 20 times lower than expected from the ionic strength of the solution and is roughly equivalent to the conductivity value of 5 mM sodium phosphate or sodium chloride solution. Table 1 shows the conductivity measurements of a number of transport buffers.

Solution / Buffer Measure 1 Measure 2 Metric 3 Mean / standard deviation 10 mM 1.95 mS / cm 2.02 mS / cm 2.13 mS / cm 2.03 +/- 0.09 mS / cm 1 mM MgCl ₂ 174 μS / cm 208 μS / cm 177 μS / cm 186 +/- 18.8 μS / cm 0.1 mM MgCl ₂ 16.9 μS / cm 16.7 μS / cm 18.3 μS / cm 17.3 +/- 0.87 μS / cm 10 mM NaCl 1.07 mS / cm 1.10 mS / cm 1.18 mS / cm 1.12 +/- 0.057 mS / cm 1 mM NaCl 112 μS / cm 115 μS / cm 111 μS / cm 112.7 +/- 2.08 μS / cm 0.1 mM NaCl 8.80 μS / cm 8.98 μS / cm 10.5 μS / cm 9.43 +/- 0.93 μS / cm 20 mM NaPO ₄ 2.90 mS / cm 2.79 mS / cm 3.00 mS / cm 2.90 +/- 0.11 mS / cm 10 mM NaPO ₄ 1.40 mS / cm 1.44 mS / cm 1.48 mS / cm 1.44 +/- 0.04 mS / cm 1 mM NaPO ₄ 122 μS / cm 128 μS / cm 136 μS / cm 128.7 +/- 7.0 μS / cm 50 mM Tris 3.50 mS / cm 3.14 mS / cm 3.40 mS / cm 3.35 +/- 0.19 mS / cm 10 mM Tris 572 μS / cm 562 μS / cm 583 μS / cm 572 +/- 10.5 μS / cm 250 mM HEPES 141 μS / cm 144 μS / cm 158 μS / cm 147.6 +/- 9.07 μS / cm 25 mM HEPES 9.16 μS / cm 9.44 μS / cm 10.5 μS / cm 9.7 +/- 0.71 μS / cm 3.3 mM sodium citrate 964 μS / cm 964 μS / cm 1.03 mS / cm 986 +/- 38.1 μS / cm 5 mM Sodium Succinate 1.05 mS / cm 960 μS / cm 1.01 mS / cm 1.01 +/- 0.045 mS / cm 5 mM Sodium Oxalate 1.02 mS / cm 1.03 mS / cm 1.12 mS / cm 1.06 +/- 0.055 mS / cm 10 mM sodium acetate 901 μS / cm 917 μS / cm 983 μS / cm 934 +/- 43.5 μS / cm 250 mM cysteine 27.4 μS / cm 17.3 μS / cm 23.5 μS / cm 22.7 +/- 5.09 μS / cm Milli-Q Water <0.5 μS / cm Too low for detection limit of 0.1 cell

Zwitterion Buffer / Conductivity / Transport Speed

The use of zwitterion buffers (excellent buffers, amino acid buffers) or tris-borate buffers in the vicinity of pi or pi has several advantages in terms of the rate or speed of transport of DNA by electrophoresis. The advantage is that 1) these buffers can be used in relatively high concentrations to increase their buffering capacity, 2) the conductivity of these buffers is much lower than other forms of buffers at the same concentration, and 3) electrophoretic for the desired analyte (DNA). The speed of transport due to the movement is faster.

Buffer capacity of zwitterion buffer at isoelectric point (pI)

Amino acid buffers also have buffer properties in their pi. A given amino acid may or may not show "best buffering capacity" in its pi but will have some buffering capacity. The buffering capacity decreases tenfold for every pH unit difference between pI and pKa, and amino acids with three ionizable groups (histidine, cysteine, lysine, glutamic acid, aspartic acid, etc.) generally have two dissociating amino acids (glycine) , Alanine, leucine, and the like) have a higher buffering capacity at pi. For example, histidine with pI = 7.47, lysine with pI = 9.74, and glutamic acid with pI = 3.22 all have relatively good buffering capacity compared to alanine or glycine, which have a relatively low buffering capacity at pI (of AL Lehninger). See Biochemistry, 2nd edition, in particular FIGS. 4-8 on page 79 and FIGS. 4-9 on page 80, Worth Publishers, New York, USA, 1975). Histidine has been proposed as a buffer for use in gel electrophoresis (see, eg, US Pat. No. 4,936,963), but no hybridization was performed in this system. Cysteine is in an intermediate position in terms of buffering capacity. The pI of cysteine is 5.02, the pKa of the alpha carboxyl group is 1.71, the pKa of the sulfhydryl group is 8.33, and the pKa of the alpha amino group is 10.78. An acid / base titration curve of 250 mM cysteine shows that the cysteine is "buffered" better than 20 mM sodium phosphate at about pH 5. In the pH range 4 to 6 the cysteine's buffering capacity is significantly better than 20 mM sodium phosphate, especially at higher pH. However, the conductivity of the 250 mM cysteine solution in the pH range is very low, about 23 μS / cm, which is 100 times smaller compared to the conductivity value of 20 mM sodium phosphate, about 2.9 mS / cm. 1 shows the conductivity measurements of various transport buffers.

Several electrophoretic techniques, developed over 20 years ago, are based on the ability to separate proteins from zwitterion buffers, "their pI state." This technique is called isophoresis, isophoresis, and isoelectric fractional electrophoresis (BD Hames and D. Rickwood, edited by Gel Electrophoresis of Proteins: A Practical Approach, lessons 3 and 4). And IRL Press, 1981). Both various amino acid buffers and “good buffers” were used in the method at their pi (see Table 2 on page 168 above).

DNA transport in buffers with low ionic strength and conductivity

A series of fluorescent checkerboard experiments were performed using a 5580 chip coated with 2.5% agarose and a ByTr-RCA5 fluorescent probe. We obtained rapid (6 sec) checkerboard addressing for both (1) 250 mM HEPES (low conductivity), (2) 10 μM sodium succinate, (3) 10 μM sodium citrate, and (4) distilled water systems. Could. The results for sodium citrate are shown in FIG. 1. Even though the properties of some types of solutions with low conductivity or ionic strength are somewhat better, checkerboard addressing and rapid DNA transport (6-12 seconds of DNA accumulation on 80 μm pads) were achieved using all of the above systems. In addition, DNA addressing APEX chips in distilled water are possible because the DNA (which itself is polyanion) is an electrolyte that provides conductivity, which is present in the bulk solution. 1 shows a plan view of an APEX chip using histidine.

Relationship between electrophoretic transport rate and cation / anion flow

In addition to the fact that the mobility of charged analytes (DNA, proteins, etc.) is related to the ionic strength of the electrolyte solution, it can be greatly influenced by the nature of the cations and anions in the electrolyte solution (see "Capillary Electrophoresis: Principles and See page 89 in "Practice". This particular point in DNA transport has been demonstrated in the above-mentioned Biopolymers, Vol. 2, pp. 231-236, 1964. 1 on page 232 of this reference shows that DNA mobility changes when using electrolytes containing different monovalent anions of the same ionic strength (Li ⁺ > Na ⁺ > K ⁺ > TMA ⁺ ). Basically different cations can have different binding constants for DNA phosphate groups and / or change the hydration region around the DNA molecule, thus changing the rate of transport.

The present invention relates to various parameters, electrolytes (buffers), and other conditions that enhance or optimize DNA transport rates, efficiency of DNA hybridization reactions, and overall hybridization specificity in molecular biological electric field devices, particularly APEX microelectronic chips and devices. It is related to the findings of the inventors. In particular, the present invention is directed to low-conductivity zwitterion buffers containing histidine amino acids prepared at concentrations of 10 to 100 mM for both rapid DNA transport and efficient hybridization by electrophoresis at or near pI (isoelectric point = about 7.47) or pi. The present invention relates to the findings of the present inventors to provide an optimum condition for the This advantage of histidine buffer is particularly important for APEX chip type devices. Such special devices (as opposed to micromechanical devices) have limitations in terms of the amount of current and voltage they can apply. Because of this limitation, it is difficult to achieve both rapid transport and efficient hybridization using one buffer system. In this case DNA transport was performed in a low conductivity buffer (cysteine or alanine), where DNA transport is still rapid even with limited current / voltage. Under these conditions DNA accumulated at the test site but did not hybridize efficiently. After transport in the low conductivity buffer, the solution was replaced with a high salt concentration buffer (> 100 mM sodium chloride or sodium phosphate) to efficiently hybridize DNA at the test site.

Table 2 shows the results of a series of experiments that correlate parameters such as buffer capacity, pH, and conductivity with DNA accumulation and hybridization sensitivity (efficiency) using the APEX chip device.

solution Buffer dose pH 4-10 pH at pI Conductivity (μS) Relative speed of DNA transport SA-Biotin T12 Sensitivity Hybridization Sensitivity of DNA β-alanine pK ₁ -3.6 pK ₂ -10.2 + 7.3 10.0 +++++ (fastest) 3 x 10 ⁶ Taurine pK ₁ -1.5 pK ₂ -8.7 +/- 4.6 4.5 ++++ > 7.5 x 10 ¹⁰ Cysteine pK ₁ -1.7 pK ₂ -8.3 pK ₃ -10.8 +/- 5.2 25.0 ++++ 3 x 10 ⁷ 7.5 x 10 ¹⁰ Histidine pK ₁ -1.8 pK ₂ -6.0 pK ₃ -9.0 +++ 7.6 212.0 (172.0 if high purity) +++ 3 x 10 ⁶ 3 x 10 ⁶ Lysine pK ₁ -2.2 pK ₂ -8.9 pK ₃ -10.3 ++ 9.6 477.0 ++ > 7.5 x 10 ¹⁰ NaPO ₄ Complex + 7.4 ^{1 /} 1,400.0 + (Slowest)

1 / is 20 mM NaPO _{4 set} to pH 7.4
In particular, Table 2 shows the various zwitterionic amino acid buffers [β-alanine, taurine, cysteine, histidine, lysine, and sodium phosphate (Zwitterionic buffer) Not)). In terms of transport, conductivity is largely related to transport under the same electric field conditions. β-alanine, taurine and cysteine show excellent transport, histidine shows good transport, and lysine and NaPO ₄ show normal transport. DNA hybridization sensitivity is for “normal DNA” having a negatively charged polyanionic phosphate backbone. Table 2 also reports the sensitivity of streptavidin / biotin DNA probe capture affinity in addition to hybridization sensitivity.

Table 2 clearly shows the correlation between DNA transport (accumulation) and low conductivity (β-alanine, taurine, cysteine, histidine). Table 2 shows that the sensitivity is excellent for streptavidin / biotin probe affinity when using β-alanine, cysteine, and histidine. As reflected in the sensitivity data in Table 2, the hybridization efficiency of histidine is at least 10 ⁴ better than cysteine or other buffers such as 20 mM NaPO ₄ . Compared to cysteine, the hybridization efficiency was improved by at least 10 times, more specifically at least 10 ² and most particularly at least 10 ⁴ . Most importantly, Table 2 shows that the DNA hybridization sensitivity (efficiency) of histidine buffer is very good. Therefore, of all the zwitterionic amino acid buffers currently tested, only histidine has good transport rate and good DNA / DNA hybridization efficiency.

Histidine buffer systems are believed to have a rapid DNA transport (accumulation) because of their low conductivity. Various explanations may be made as to why DNA / DNA hybridization is relatively efficient in histidine buffer. The good buffering capacity of histidine may be an advantage. Histidine will buffer well under acidic or basic conditions at a pI of 7.47 (see Lenninger Biochemistry, 2nd edition, pages 4-9, 1975, Worth Publishers, NY, 1975). ). APEX chips generate acid at the anode where DNA accumulates for hybridization, and histidine can effectively buffer these conditions. More importantly, under the acidic conditions (pH <5), the histidine molecule begins to be converted to a divalent cationic species by protonation of the imidazole group of histidine. The divalent cations having a positively charged α-amino group and a positively charged imidazole group may assist in promoting hybridization at the anode of the APEX chip and stabilizing the formed DNA / DNA hybrid. Cations, divalent cations, and polyvalent cations are known to help stabilize DNA / DNA hybrids by reducing the repulsion of the negatively charged phosphate backbone of double stranded DNA constructs. DNA / DNA / histidine may also form some form of stabilizing adduct from other electrochemical products (such as hydrogen peroxide) produced at the anode.

In the present embodiment, natural histidine is used, but in the present invention, other natural or synthetic compounds having excellent buffering ability and low conductivity (or having zwitterion characteristics) and stabilizing DNA hybridization by charge stabilization or adduct formation. Compounds can also be used.

Although the present invention has been described in detail with examples and examples for clarity and understanding of the present invention, those skilled in the art, in view of the teachings of the present invention, may change the present invention without departing from the spirit or scope of the appended claims. And can be changed.

Claims (55)

(1) (a) low conductivity, (b) the original isoelectric point (pI) is higher than 3.5, (c) have an effective buffering capacity substantially between pH 4 and pH 10 Adding a buffer, selected according to the criteria, into the device, (2) (a) is sufficient to generate an electric field at the test site such that the test site of the microelectronic device is positively biased relative to the DNA analyte; (b) provide a net positive charge to the buffer molecule under the influence of the electric field so that the positively charged buffer molecule is sufficient to act to stabilize the DNA hybridization between the DNA analyte and the single stranded capture DNA immobilized at the test site. Applying a positive current to the test site of the microelectronic device A method of electronically enhancing hybridization of a DNA analyte to a single stranded capture DNA immobilized at a test site under the influence of an electric field at a test site biased in the amount of microelectronic device. The method of claim 1, wherein the low conductivity buffer is a zwitterion buffer. The method of claim 2, wherein the zwitterion buffer contains histidine. The method of claim 3, wherein the histidine is prepared at a concentration of about 10-100 mM. The method of claim 3 wherein the pH of the histidine is prepared at or near isoelectric point. The method of claim 1 wherein the pH of the isoelectric point is about 7.47. The method of claim 1, wherein the buffer stabilizes hybridization between the target nucleic acid and the capture nucleic acid. 8. The method of claim 7, wherein the buffer is a natural compound with low conductivity. 8. The method of claim 7, wherein the buffer is a zwitterionic natural compound. 8. The method of claim 7, wherein the buffer is a synthetic compound with low conductivity. 8. The method of claim 7, wherein the buffer is a zwitterionic synthetic compound. The method of claim 1 wherein the hybridization efficiency is at least 100 times greater than for cysteine under the same conditions. The method of claim 1 wherein the hybridization efficiency is at least 1000 times greater than for cysteine under the same conditions. The method of claim 1 wherein the hybridization efficiency is at least about 50000 times greater than for cysteine under the same conditions. The method of claim 1, wherein the buffer reduces the repulsive action between the capture nucleic acid and the target nucleic acid. The method of claim 1, wherein the buffer reduces adduct formation between the capture nucleic acid and the target nucleic acid. In a microelectron hybridization device comprising a microcompartment test site having a capture nucleic acid, the microcompart can be at least in a first state in which no electric field is generated and in a second state in which an electric field is attracted to the target nucleic acid in the microcompartment. As a method of enhancing the hybridization efficiency of a target nucleic acid, Putting the buffer into the device, Providing a target nucleic acid to the device, Applying power to the device to bring the microcompartments into the second state such that target nucleic acids accumulate at the microcompartment test site of the device, and Hybridizing target nucleic acid and capture nucleic acid with greater efficiency when in the second state than in the first state How to include. delete delete delete delete delete delete delete delete delete delete delete 18. The method of claim 17, wherein the hybridization efficiency is at least 1000 times greater than for cysteine under the same conditions. 18. The method of claim 17, wherein the hybridization efficiency is at least about 50000 times greater than for cysteine under the same conditions. The method of claim 17, wherein the buffer reduces the repulsive action between the capture nucleic acid and the target nucleic acid in the second state. The method of claim 17, wherein the buffer reduces adduct formation between the capture nucleic acid and the target nucleic acid. The method of claim 1 wherein the low conductivity is substantially less than 1 mS. The method of claim 1 wherein the low conductivity is substantially less than 500 μs. The method of claim 1 wherein the low conductivity is substantially less than 250 μs. The method of claim 1 wherein the low conductivity is substantially less than 100 μs. The method of claim 1, wherein the original pi of the buffer is substantially 7.4 or greater. The method of claim 1, wherein the buffer molecule protects the analyte from hydrogen generated at the positively biased test site by providing a buffering effect to the solution around the test site. The method of claim 1 wherein the buffer molecule with a net positive charge is a cation. The method of claim 1 wherein the buffer molecule with a net positive charge is a divalent cation. The method of claim 1 wherein the buffer molecule with a net positive charge is a polyvalent cation. The method of claim 1, wherein the buffer is selected such that, in the absence of an electric field, the DNA fails to shield substantially the DNA to aid in DNA hybridization. The method of claim 17, wherein the buffer is low in conductivity. delete The method of claim 43, wherein the low conductivity buffer is a zwitterion buffer. 46. The method of claim 45, wherein the zwitterion buffer contains histidine. The method of claim 46, wherein the histidine is prepared at a concentration of about 10-100 mM. 47. The method of claim 46, wherein the histidine pH is prepared at or near isoelectric point. The method of claim 43, wherein the pH of the isoelectric point is about 7.47. The method of claim 43, wherein the buffer stabilizes hybridization between the target nucleic acid and the capture nucleic acid in the second state. 51. The method of claim 50, wherein the buffer is a natural compound with low conductivity. 51. The method of claim 50, wherein the buffer is a zwitterionic natural compound. 51. The method of claim 50, wherein the buffer is a synthetic compound with low conductivity. 51. The method of claim 50, wherein the buffer is a zwitterionic synthetic compound. 44. The method of claim 43, wherein the hybridization efficiency is at least 100 times greater than for cysteine under the same conditions.

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