METHODS AND MATERIALS FOR OPTIMIZATION OF ELECTRONIC HYBRIDIZATION REACTIONS

PROCEDES ET MATERIELS POUR OPTIMISER DES REACTIONS D'HYBRIDATION ELECTRONIQUES

English Abstract

The following inventions relate to discoveries concerning the various
parameters, electrolytes (buffers), and other conditions which
improve or optimize the speed of DNA transport, the efficiency of DNA
hybridization reactions, and the overall hybridization specificity in
microelectronic chips and devices. In particular, this invention relates to
the discovery that low conductance zwitterionic buffer solutions,
especially those containing the amino acid Histidine prepared at
concentrations of ~50 mM and at or near the pI (isoelectric point ~pH 7.47),
provide optimal conditions for both rapid electrophoretic DNA transport and
efficient hybridization reactions. Hybridization efficiencies of
at least a factor of 10 relative to the next best known buffer, Cysteine, are
achieved. Test data demonstrate an approximately 50,000 fold
increase in hybridization efficiency compared to Cysteine.

French Abstract

La présente invention concerne des découvertes se rapportant aux divers paramètres, électrolytes (tampons) et autres conditions permettant d'améliorer ou d'optimiser la vitesse de déplacement de l'ADN, le rendement des réactions d'hybridation d'ADN et la spécificité d'hybridation globale de puces et dispositifs microélectroniques. L'invention concerne en particulier la découverte selon laquelle des solutions tampons zwittérioniques de faible conductance, notamment celles contenant l'aminoacide histidine préparé à des concentrations de SIMILAR 50 mM et à un point pHi ou proche du pHi (point isoélectrique SIMILAR pH 7,47), offrent des conditions optimales pour un transfert électrophorétique rapide de l'ADN et des réactions d'hybridation efficaces. Des rendements d'hybridation d'au moins un facteur de 10 par rapport au meilleur tampon connu après celui de l'invention, la cystéine, sont obtenus. Des données d'essai montrent une efficience d'hybridation augmentée d'environ 50 000 fois, comparée au résultat de la cystéine.

Claims

14
CLAIMS:

1. A method for transporting and hybridizing target
nucleic acids in a microelectronic device having at least
one test site bearing a capture nucleic acid, comprising the
steps of:
(1) applying a histidine buffer to the device,
wherein the histidine buffer has a low conductance of less
than 1 mS,
(2) applying current to the device to produce an
electric field at the test site,
(3) transporting the target nucleic acids to the
test site, and
(4) hybridizing the target nucleic acids to the
capture nucleic acid at the test site with a hybridization
efficiency which is at least a factor of 10 times greater
than for cysteine buffer under the same conditions.

2. The method of claim 1 wherein the histidine buffer
is selected according to the following criteria:
(a) the buffer having low conductance of less than
1 mS,
(b) the buffer having an isoelectric point (pI)
above 3.5, and
(c) the buffer having effective buffering capacity
between pH 4 and pH 10.

3. The method of claim 1 or 2, wherein the histidine
buffer was prepared at a concentration of about 10-100 mM.





15

4. The method of any one of claims 1 to 3, wherein
the histidine buffer having an isoelectric point was
prepared at or about the isoelectric point.

5. The method of claim 4, wherein the isoelectric
point is about 7.47.

6. The method of any one of claims 1 to 5, wherein
the histidine buffer stabilizes hybridization between the
target nucleic acids and the capture nucleic acid.

7. The method of any one of claims 1 to 6, wherein
the histidine is a natural, zwitterionic compound.

8. The method of any one of claims 1 to 6, wherein
the histidine is a synthetic, zwitterionic compound.

9. The method of any one of claims 1 to 8, wherein
the histidine buffer has a low conductance of less
than 500 µS.

10. The method of any one of claims 1 to 8, wherein
the histidine buffer has a low conductance of less than
250 µS.

11. The method of any one of claims 1 to 8, wherein
the histidine buffer has a low conductance of less than
100 µS.

12. The method of any one of claims 1 to 11, wherein
the histidine buffer has a pI above 7.4.

13. The method of any one of claims 1 to 12, wherein
the histidine buffer reduces repulsion between the capture
nucleic acid and the target nucleic acids.





16

14. The method of any one of claims 1 to 13, wherein
the histidine buffer reduces adduct formation between the
capture nucleic acid and the target nucleic acids.

15. The method of any one of claims 1 to 14, wherein
the hybridization efficiency is at least a factor of 100
times greater than for cysteine buffer under the same
conditions.

16. The method of any one of claims 1 to 15, wherein
the hybridization efficiency is at least a factor of 1,000
times greater than for cysteine buffer under the same
conditions.

17. The method of any one of claims 1 to 16, wherein
the hybridization efficiency is at least a factor of 50,000
times greater than for cysteine buffer under the same
conditions.

18. A method for enhancing the hybridization
efficiency of target nucleic acids in a microelectronic
hybridization device including a microlocation test site
having a capture nucleic acid, the microlocation adapted to
be placed at least in a first state wherein no electric
field is generated and in a second state wherein an electric
field attractive to the target nucleic acids is generated at
the microlocation, comprising the steps of:
applying a histidine buffer to the device,
providing target nucleic acid to the device,
placing the microlocation in the second state
through application of power to the device to cause
accumulation of the target nucleic acids at a microlocation
test site on the device, and




17

hybridizing the target nucleic acids with the
capture nucleic acid with an efficiency which is greater in
the second state than in the first state.

19. The method of claim 18, wherein the histidine
buffer has a low conductance of less than 1 mS.

20. The method of claim 18 or 19, wherein the
histidine buffer was prepared at a concentration of about
10-100 mM.

21. The method of any one of claims 18 to 20, wherein
the histidine buffer was prepared at or about the
isoelectric point.

22. The method of claim 21, wherein the isoelectric
point is about 7.47.

23. The method of any one of claims 18 to 22, wherein
the histidine buffer stabilizes hybridization between the
target nucleic acids and the capture nucleic acid in the
second state.

24. The method of any one of claims 18 to 23, wherein
the histidine is a natural compound with low conductance of
less than 1 mS.

25. The method of any one of claims 18 to 23, wherein
the histidine is a natural, zwitterionic compound.

26. The method of any one of claims 18 to 23, wherein
the histidine is a synthetic compound with low conductance
of less than 1 mS.

27. The method of any one of claims 18 to 23, wherein
the histidine is a synthetic, zwitterionic compound.




18

28. The method of any one of claims 18 to 27, wherein
the histidine buffer has a low conductance of less than
500 µS.

29. The method of any one of claims 18 to 27, wherein
the histidine buffer has a low conductance of less than
250 µS.

30. The method of any one of claims 18 to 27, wherein
the histidine buffer has a low conductance of less than
100 µS.

31. The method of any one of claims 18 to 30, wherein
the histidine buffer has a pI above 7.4.

32. The method of any one of claims 18 to 31, wherein
the hybridization efficiency is at least a factor of 100
times greater than for cysteine buffer under the same
conditions.

33. The method of any one of claims 18 to 32, wherein
the hybridization efficiency is at least a factor of 1,000
times greater than for cysteine buffer under the same
conditions.

34. The method of any one of claims 18 to 33, wherein
the hybridization efficiency is at least a factor of 50,000
times greater than for cysteine buffer under the same
conditions.

35. The method of any one of claims 18 to 34, wherein
the histidine buffer reduces repulsion between the capture
nucleic acid and the target nucleic acids in the second
state.




19

36. The method of any one of claims 18 to 35, wherein
the histidine buffer reduces adduct formation between the
capture nucleic acid and the target nucleic acids.

37. A method for electronically enhancing
hybridization of DNA analyte to single stranded capture DNA
bound at a test site in an electric field at a positively
biased test site on a microelectronic device, comprising the
steps of:
(1) applying a histidine buffer to the device, the buffer
being selected according to the following criteria:
(a) the histidine buffer having a low conductance
of less than 1 mS,
(b) the histidine buffer having an isoelectric
point (pI) above 3.5, and
(c) the histidine buffer having effective
buffering capacity between pH 4 and pH 10, and
(2) applying current to the test site on the microelectronic
device in an amount sufficient to:
(a) produce an electric field at the test site on
the microelectronic device, the test site being positively
biased relative to the DNA analyte, and
(b) provide a net positive charge on the buffer
molecules under influence of the electric field, the
positively charged buffer molecules serving to stabilize DNA
hybridization between the DNA analyte and the single
stranded capture DNA bound at the test site.

38. The method of claim 37, wherein the histidine
buffer was prepared at a concentration of about 10-100 mM.




20

39. The method of claim 37 or 38, wherein the
histidine buffer was prepared at or about the isoelectric
point.

40. The method of claim 39, wherein the isoelectric
point is about 7.47.

41. The method of any one of claims 37 to 40, wherein
the histidine buffer stabilizes hybridization between the
target nucleic acids and the capture nucleic acid.

42. The method of any one of claims 37 to 41, wherein
the histidine buffer is a natural compound with low
conductance.

43. The method of any one of claims 37 to 41, wherein
the histidine buffer is a natural, zwitterionic compound.

44. The method of any one of claims 37 to 41, wherein
the histidine buffer is a synthetic compound with low
conductance.

45. The method of any one of claims 37 to 41, wherein
the histidine buffer is a synthetic, zwitterionic compound.

46. The method of any one of claims 37 to 45, wherein
the histidine buffer reduces repulsion between the capture
nucleic acid and the target nucleic acids.

47. The method of any one of claims 37 to 46, wherein
the histidine buffer reduces adduct formation between the
capture nucleic acid and the target nucleic acids.

48. The method of any one of claims 37 to 47, wherein
the histidine buffer has a low conductance of less than
500 µS.





21

49. The method of any one of claims 37 to 47, wherein
the histidine buffer has a low conductance of less than
250 µS.

50. The method of any one of claims 37 to 47, wherein
the histidine buffer has a low conductance of less than
100 µS.

51. The method of any one of claims 37 to 50, wherein
the histidine buffer has a pI above 7.4.

52. The method of any one of claims 37 to 51, wherein
the histidine buffer molecules provide protection to the
analyte from the hydrogen generated at the positively biased
test site by buffering the solution around the test site.

53. The method of claim 37, wherein the histidine
buffer molecules having a net positive charge are cationic.

54. The method of claim 37, wherein the histidine
buffer molecules having a net positive charge are
dicationic.

55. The method of claim 37, wherein the histidine
buffer molecules having a net positive charge are
polycationic.

56. The method of any one of claims 37 to 55, wherein
the histidine buffer is selected such that it is
substantially incapable of shielding DNA and therefore not
supportive of DNA hybridization in the absence of an
electric field.

57. The method of any one of claims 1 to 16, wherein
the hybridization efficiency is at least a factor of 25,000
times greater than for cysteine buffer under the same
conditions.





22

58. The method of any one of claims 18 to 31, wherein
the hybridization efficiency is at least a factor of 10
times greater than for cysteine buffer under the same
conditions.

59. The method of any one of claims 18 to 33, wherein
the hybridization efficiency is at least a factor of 25,000
times greater than for cysteine buffer under the same
conditions.

Description

1015202530CA 02264780 2003-O5-O550338-141DESCRIPTIONMethods And Materials For OptimizationOf Electronic Hybridization ReactionsField of the InventionThis invention relates to buffers and electrolytesfor use in electronic devices adapted for medicaldiagnostic, biologicalgand other uses. More particularly,it relates to buffers and electrolytes for advantageoususe with DNA hybridizationanalysis carried outonmicroelectronic medical diagnostic devices.Background of the InventionRecently, there has been increasing interest indevices which combine microelectronics and molecularbiology. One such system is disclosed in "ACTIVEPROGRAMMABLE ELECTRONIC DEVICES FOR MOLECULAR BIOLOGICALANALYSIS AND DIAGNOSTICS", 08/146,504, filed1993, now issued- as United States Patent5,605,662. Thesystems disclosed therein will be referred to as APEXsystems.Serial No.November 1,No.AEEX systems are able to perform a wide varietyof functions which are advantageously used in molecularbiology reactions, such as nucleic acid hybridizations,antibody/antigen reactions, clinical diagnostics, andbiopolymer synthesis.APEX-type devices utilize buffers and electrolytesfor their operation. A buffer has been defined as achemical solution which is resistant to change in pH onthe addition of acid or alkali. See., e.g., Dictionary ofCoombs, Stocktonbuffers based onBiotechnology, Second Edition,As stated there,JamesPress. "traditionally,inorganic salts (phosphate, carbonate) and organic acidsalts (acetate, citrate, succinate, glycine,barbiturates, etc.) were used in biological experiments."maleate,101.520253 0CA 02264780 2004-02-0450338-142It is the object of this invention to discoverbuffers and electrolytes which are advantageously used inmolecular biology electronic devices which performreactions,hybridizations, diagnostics or synthesis.Summary of the InventionThe following inventions relate to our discoveriesconcerning the various parameters, electrolytes (buffers),and other conditions which improve or optimize the speed ofDNA transport, the efficiency of DNA hybridizationreactions, and the overall hybridization specificity in ourAPEX microelectronic chips and devices. In particular, thisinvention relates to the discovery that low conductancezwitterionic buffer solutions, especially those containingthe amino acid Histidine prepared at concentrations of10-100 mM, preferably about 50 mM, and at or near the pI(isoelectric point ~pH 7.47), provide optimal conditions forboth rapid DNA transport and efficient hybridizationreactions. Hybridization efficiencies of at least a factorof 10 relative to the next best known buffer, Cysteine, areachieved. Test data demonstrate an approximately 50,000fold increase in hybridization efficiency compared toCysteine.In one aspect the invention relates to a methodfor transporting and hybridizing target nucleic acids in amicroelectronic device having at least one test site bearinga capture nucleic acid, comprising the steps of:(1) applying a histidine buffer to the device, wherein thehistidine buffer has a low conductance of less than 1 mS,(2) applying current to the device to produce an electricfield at the test site, (3) transporting the target nucleicacids to the test site, and (4) hybridizing the targetnucleic acids to the capture nucleic acid at the test site1015202530CA 02264780 2004-02-0450338-142awith a hybridization efficiency which is at least a factorof 10 times greater than for cysteine buffer under the sameconditions.In another aspect the invention relates to amethod for enhancing the hybridization efficiency of targetnucleic acids in a microelectronic hybridization deviceincluding a microlocation test site having a capture nucleicacid, the microlocation adapted to be placed at least in afirst state wherein no electric field is generated and in asecond state wherein an electric field attractive to thetarget nucleic acids is generated at the microlocation,comprising the steps of: applying a histidine buffer to thedevice, providing target nucleic acid to the device, placingthe microlocation in the second state through application ofpower to the device to cause accumulation of the targetnucleic acids at a microlocation test site on the device,and hybridizing the target nucleic acids with the capturenucleic acid with an efficiency which is greater in thesecond state than in the first state.In another aspect the invention relates to amethod for electronically enhancing hybridization of DNAanalyte to single stranded capture DNA bound at a test sitein an electric field at a positively biased test site on amicroelectronic device, comprising the steps of: (1)applying a histidine buffer to the device, the buffer beingselected according to the following criteria: (a) thehistidine buffer having a low conductance of less than 1 ms,(pl)and (c) the histidine buffer having effective(b) the histidine buffer having an isoelectric pointabove 3.5,buffering capacity between pH 4 and pH 10, and (2) applyingcurrent to the test site on the microelectronic device in anamount sufficient to: (a) produce an electric field at thetest site on the microelectronic device, the test site being1.01.5CA 02264780 2004-02-0450338-14213positively biased relative to the DNA analyte, and (b)provide a net positive charge on the buffer molecules underinfluence of the electric field, the positively chargedbuffer molecules serving to stabilize DNA hybridizationbetween the DNA analyte and the single stranded capture DNAbound at the test site.Brief Description of the DrawingsFig. 1 is a plan view of a checkerboardarrangement utilizing a histidine buffer.Detailed Description of the InventionThere are various physical parameters which relateto the electrophoretic transport of DNA and other chargedanalytes in various types of electrolyte/buffer solutions.Certain of the devices, e.g., Applicant's APEX device asdescribed in United States Patent No. 5,605,662, referencedabove, are basically DC (direct current) electrical deviceswhich generate electric fields on the surface of the device.These fields,in turn, cause the101520253035W0 98/10273CA 02264780 1999-03-01PCT/U S97/ 144893electrophoretic transport of charged molecules to occur(+/-)By contrast the so—called Genosensorbetween oppositely biased. microlocations on thedevice surface.(impedance sensors), see, e.g., Hollis et al, "Optical andElectrical Methods and Apparatus for Molecular Detection",W093/22678, devices, see, e.g.,Washizu 25 Journal of Electrostatics, 109-123, 1990,An importantand dielectrophoresisinvolve the use of .AC electric fields.distinction related to these devices is that when these ACfields are applied, there is essentially no net currentflow in any of these systems, i.e, there is no electropho-retic propulsion for transport of the charged molecules.APEX type devices produce significant net direct current(DC) flow when a voltage is applied, which is recognizedas "the signature of electrophoresis". In electrophore-sis, the migration of ions or charged particles isproduced by electrical forces along the direction of theelectric field gradient, and the relationship of currentand voltage are important to this technology. Theelectrophoretic migration shows itself macroscopically asthe conduction of electric current in a solution under theinfluence of an applied voltage and follows Ohm’s law:V=RxIV is the electric potentialR is the electric resistance of the electrolyte [VxA‘1=R(§2)]I is the electric current [A].The resistance of the solution is the reciprocal ofthe conductance which can be measured by a conductometer.The conductance depends mainly on the ionic species of thebuffer/electrolytes and their concentration; thereforethese parameters are very important for electric fieldrelated molecular biology technology. The basiccurrent/voltage relationships are essentially the same forthe APEX technology assystem, although the electric fields produced are in trulyfor any other electrophoreticmicroscopic environments.101520253035W0 98/ 10273CA 02264780 1999-03-01PCT/U S97/ 144894APEXregarding the various ways of sourcing the current andThere are unique features of the systemvoltage, and how the current and voltage scenarios havebeen found to improve the performance of such systems. In(linear andparticular, various DC pulsing procedureslogarithmic gradients) appear to provide improvedhybridization stringency.Electrophoretic Transport Versus Ionic StrengthIt is well established in the field of electro-phoresis that there is a logarithmic decrease in themobility of the charged analyte species (proteins, DNA,etc.), which is inversely proportional to the square rootof the ionic strength of the electrolyte solution (seepage 83 and Fig. 3.16 in "Capillary Electrophoresis:Kuhn and S. Hoffstetter,At any given constant electricPrinciples and Practice", R.Springer—Verlag, 1993).field strength, as the electrolyte concentration decreasesrelative to the analyte species (protein, DNA, etc.), theanalyte will be transported at a faster rate. Similarresults demonstrating this effect for a danyslated aminoacid have been shown by J.J. Issaq et. al.,Chromatographia Vol. 32, #3/4, August 1991, pages 155 to161 (see iJ1 particular Fig. 13 on page 157). Resultsdemonstrating this effect for DNA is different electrolytesolutions has been shown in P.D. Ross and R.L. Scruggs,pages 231 to 236, 1964 (see inparticular Fig. 1, page 232).Biopolymers Vol. 2,Ionic Strengthzconductance RelationshipFor those non—buffering electrolytes (sodiumchloride, potassium chloride, etc.) which involvecompletely dissociated anion and cation species insolution (Na* <-—-> Cl‘, K* <-—-> Cl‘, etc.), the ionicstrength and conductance are equivalent, i.e., theconductance will "usually’ be proportional to the ionicstrength. For those buffering electrolytes (phosphate,l01520253035W0 98/10273:strength or concentration.CA 02264780 1999-03-015which are in their2 Na‘ <-——> PO4"2), the ionicstrength and conductance will usually be equivalent, i.e.,acetate, citrate, succinate, etc.)dissociated states (example:conductance is proportional to the ionic strength. For(MOPS , HEPES ,Ampholytes,those buffering electrolytes [Good BuffersTAPS ,etc.] which can have a zwitterionic species (no net chargeTricine, Bicine), Amino Acid Buffers,at their pI), the conductance will decrease by approxi-mately a factor of 10 for every pH unit difference betweenthe isoelectric point (pl) and the (pKa). For example, anamino acid in its zwitterionic state VOOC—CH(R)—NH{) willhave a conductance value which will be approximately 1000fold lower than when the "amino acid moiety" has a full(HOOC-CH(R)-NH2’ <———> X’) ,negative charge (Y* <—-->' OOC-CH(R)—NH2). Thus,negative or positive charge develops on the amino acidnet positive charge or a fulla formalmoiety as it moves away from its pI, and the conductivityand ionic strength will begin to correlate. However, whenat or near the pI the conductance will be much lower thanis expected for that given ionic strength or concentra-tion. When used at or near their pI's, electrophoresistexts refer to the Good Buffers and amino acid buffers asat high(see page 88 of Capillary Electrophoresis:Kuhn and S. Hoffstetter,A commonly used electrophoresishaving "low conductances ionic strength orconcentration"Principles and Practice", R.Springer — Verlag, 1993).bufferconductivity than would be"Tris-Borate" actually has a significantly lowerexpected from_ its ionicThis may be due to the "triscation" "borate anion"and forming a relatively stablezwitterionic complex in solution. The conductivity of a100 mM Tris—Borate solution was determined to be 694us/cm, which is approximately 20 times lower than would beexpected from its ionic strength, and is roughly equiva-lent to a 5 mM sodium phosphate or sodium chloridesolution. Table i shows conductivity measurements of anumber of transport buffers.PCT/US97/14489101520253035CA02264780 1999-03-01PCT/US97/ 14489W0 98/10273Solution/B Average/Std.utter Measurement 1 Measurement 2 Measurement 3 Deviation10 mM 1.95 ms/cm 2.02 ms/cm 2.13 ms/cm 2.03+/-0.09Mgcl, ms/cm1 mM MgCl, 174 us/cm 208 us/cm 177 us/cm 186+/-18.8us/cm0.1 mM 16.9 us/cm 16.7 us/cm 18.3 us/cm 17.3+/-0.87Mgclz us/cm10 mM Nacl 1.07 ms/cm 1.10 ms/cm 1.18 ms/cm 1.12+/-0.057ms/cm1 mM NaC1 112 us/cm 115 us/cm 111 us/cm 112.7+/-2.08us/cm0.1 mM 8.80 us/cm 8.98 us/cm 10.5 us/cm 9.43+/-0.93NaCl us/cm20 mM 2.90 ms/cm 2.79 ms/cm 3.00 mS/cm 2.90+/-0.11NaPO. ms/cm10 mM 1.40 ms/cm 1.44 ms/cm 1.48 ms/cm 1.44+/-0.04NaP0, ms/cm1 mM Napo. 122 u.S/cm 128 us/cm 136 us/cm 12e.7+/-7.0us/cm50 mM TRIS 3.50 ms/cm 3.14 ms/cm 3.40 ms/cm 3.35-r/-0.19ms/cm10 mM TRIS 572 us/cm S62 us/cm 583 us/cm 572+/-10.5us/cm250 mM 141 us/cm 144 us/cm 15B MS/cm 147.6+/-9.07HEPES us/cm25 mM 9.16 us/cm 9.44 us/cm 10.5 us/cm 9.7+/-0.71HEPES us/cm3.3 mM 964 us/cm 964 us/cm 1.03 ms/cm 986+/-38.1Nacitrate us/cm5 mM 1.05 ms/cm 960 us/cm 1.01 ms/cm 1.01+/-0.045NaSucci- ms/cmnate5 mM 1.02 ms/cm 1.03 ms/cm 1.12 ms/cm 1.06+/-0.055Naoxalate ms/cm10 NM 901 #3/Cm 917 us/cm 983 [LS/cln 934+/-43.5NaAcetate us/cm250 mM 27.4 us/cm 17.3 us/cm 23.5 us/cm 22.7+/-5.09Cysteine us/cmMilli-Q <0.5 us/cm Detection limitwater of0.1 cell toolowTable 1l01520253035WO 98/10273CA 02264780 1999-03-01PCTIUS97l14489'7Zwitterionic Buffers/Conductance/Transport RateCertain advantages exist regarding the rate or speedtransport of DNA(Good buffers, amino acid buffers),or the Tris-Borate buffer at or near their pIs. These1) such buffers can be used at relatively highconcentrations to increase buffering capacity, 2)conductances are significantly lower than other types ofand 3)advantage of higher electrophoretic transport rates for(DNA).ofZwitterionic bufferselectrophoretic when usingare:theirbuffers at the same concentration, one gains thethe analyte of interestZwitterionic Buffer Capacity at the Isoelectric Point (nilAmino acid buffers do have buffer properties at theirpI’s. While a given amino acid may or may not have its"highest buffering capacity" at its pI, it will have somedegree of buffering capacity. Buffer capacity decreasesby a factor of 10 for every pH unit difference between thepI and the pKa; those amino acids with three ionizablegroups (histidine, cysteine, lysine, glutamic acid,aspartic acid, etc.) generally have higher bufferingcapacities at their pI’s than those amino acids with onlytwo dissociations (glycine, alanine, Forleucine, etc.).example, histidine p1 = 7.47, lysine pI=9.74, and glutamicacid pI=3.22, all have relatively good buffering capacityat their pIs, relative to alanine or glycine which haverelatively low buffering capacities at their pIs (see A.L.Lehninger, Biochemistry, 2ed, Worth Publishers, New York,1975; in particular Fig. 4-8 on page 79, and Fig. 4-9 onpage 80). Histidine has been proposed as a buffer for usein gel electrophoresis, see, e.g., U.S. Patent 4,936,963,but hybridization is not performed in such systems.Cysteine is in a more intermediate position, with regardthethe pKa for thesulfhydryl is 8.93, and the pKa for a amino group is10.78.to buffering capacity. The pI of cysteine is 5.02,pKa for the a carboxyl group is 1.71,An acid /base titration curve of 250 mM cysteine,WO 98/10273101520253035CA 02264780 1999-03-01PCT/US97ll44898shows that cysteine has a better "buffering capacity" atIn the pH 4 to 6range, the buffering capacity of cysteine is significantlybetter than 20 mM sodium phosphate,~ pH 5 than a 20 mM sodium phosphate.particularly at theHowever, in these pH ranges the conductance of~23 us/cm,compared to 20 mM sodium phosphate which has a value ofhigher pH.the 250 mM cysteine solution is very low~2.9 ms/cm, a factor of 100 times greater.theBuffers.Figure 1 showsConductivity Measurements of Various TransportSeveral electrophoretic techniques developed over 20years ago are based on the ability to separate proteins inzwitterionic buffers "at their pIs." These techniques arecalledElectrofocusingand"GelElectrophoresis of Proteins: A Practical Approach" Editedby B.D. Hames & D. Rickwood, IRL Press 1981).amino acid buffers and Good buffers were used for theseIsoelectrophoresis, Isotachophoresis,(see chapters 3 and 4 inVariousapplications, all at their pI’s (see Table 2, page 168 ofthe above reference).DNA Transport in Low Ionic Strength and Low ConductanceBuffersA series of fluorescent checkerboard experiments werecarried out using 2.5% agarose coated 5580 chips and theByTr-RCA5 fluorescent probe. ‘ We were able to achievecheckerboard addressing in all of the(1) 250 mM HEPES(2) 10 pM sodium succinate, (3) 10 uM sodium citrate, and(4) distilled water.shown in Figure 1.rapid (6 second)following systems: (low conductance),The results for sodium citrate areWhile, some types of low conductanceor low ionic strength solutions may have somewhat bettercharacteristics, checkerboard addressing and rapid DNAtransport (6 to 12 second DNA accumulation on an 80 pmpad) were achieved ‘using all of these systems.Additionally, DNA addressing APEX chips in distilled wateris possible because the DNA (itself a polyanion) is theWO 98/102731020253035CA 02264780 1999-03-01PCT/US97/ 144899electrolyte present in the bulk solution which providesthe conductance. Fig. 1 shows a plan view of an APEX chipusing histidine.Relationship of Electrophoretic Transport Rate and theCation[Anion SpeciesIn addition to the fact that the mobility of thecharged analyte species is relatedthemobility is also greatly influenced by the nature of the(DNA, proteins, etc.)to the ionic strength of the electrolyte solution,cation and anion species in the electrolyte solution (seepp 89 of "Capillary Electrophoresis:Thisdemonstrated for DNA transport in the above Biopolymers,Vol. 2, pp. 231-236, 1964 reference.of this reference shows the change in DNA mobility whenPrinciples andPractice" reference). particular point isFigure 1 on page 232using electrolytes with different univalent anions (Li * >Na‘>K*>TMA")different cations can have different association constantsat the same ionic strength. Basically,with the DNA phosphate groups, and/or change the hydrationspheres around the DNA molecules, which leads to a changein their transport rate.The instant invention relates to our discoveriesconcerning the various parameters, electrolytes (buffers),and other conditions which improve or optimize the speedof DNA. transport, the efficiency’ of DNA. hybridizationreactions, and the overall hybridization specificity inelectric field molecular biology devices, especially APEXthisinvention relates to our discovery that low conductancemicroelectronic chips and devices. In particular,zwitterionic buffer solutions containing the amino acidHistidine prepared at concentrations of 10-100 mM,especially about 50 mM, at or near the pI (isoelectricpoint ~7.47), provide optimal conditions for both rapidelectrophoretic DNA transport and efficient hybridizationreactions. This advantage of the Histidine buffer isparticularly important for the APEX chip type devices.—_.............._..........- .... .s............,;.....,........................., . .W0 98/ 102731015CA 02264780 1999-03-01PCT/US97/ 1448910These particular devices (as opposed to the micromachinedtype devices) have limitations as to the amount of currentand voltages that can be applied. This limitation makesit difficult to achieve both rapid transport and efficienthybridization using the same buffer system. In thesecases, DNA transport was carried out in a low conductancebuffer limitedcurrent/voltage still produced rapid transport.(Cysteine or Alanine) where theUnderthese conditions the DNA accumulated at the test site, butdid not hybridize as efficiently. After transport inthese low conductance buffers, the solution was changed toa high salt buffer (> 100 mM sodium chloride or sodiumphosphate) which then produced an efficient hybridizationat the test site.Table 2 shows the results for a series of experimentswhich correlate the parameters of buffer capacity, pH, andthe conductivity, with DNA accumulation and hybridizationsensitivity (efficiency) using the APEX chip device.CA 02264780 1999-03-01WO 98110273 PCT/US97/1448911Relative Hybridiza-pH DNA SA- tionSolution Buffer Capacity at Conduc- Transport Biotin Sensitiv-pH 4-10 PI tivity Rate T12 ity of DNA(us) sensi-tivity[3-Alanine pK, - 3.6 + 7.3 10.0 +++++ 3 x 10‘pK, - 10.2 (fastest)Taurine pK, - 1.5 +/- 4.6 4.5 ++++ > 7.5 xpx, - 3.7 10”Cysteine pK, - 1.7 +/- 5.2 25.0 ++++ 3 x 107 7.5 x 10”pK2 - 8.3pK, - 10.85 Histidine px, - 1.8 +++ 7.6 212.0 +++ 3 x 10‘ 3 x 10‘px, - 6.0 (172.0pk, - 9.0 hipurity)Lysine pK, - 2.2 ++ 9.6 477.0 H» > 7.5 xpx, - 8.9 10“13K, - 10.3Napo, Complex + 7 . 41’ 1, 400 . o +(slowest)TABLE 210152025In particular, Table 2 shows the effect of variouszwitterionic amino acid buffers [B—Alanine, Taurine,Cysteine, Histidine, Lysine, and Sodium Phosphate (not azwitterionic buffer)] on the hybridizability of thetransported target DNA to the specific capture DNA at thetest site. As to transport, the conductivity generallycorrelates with transport under the same field conditions.B—Alanine, Taurine and Cysteine show excellent transport,Histidine shows good transport, and Lysine and NaPO4 showfair transport. The DNA hybridization sensitivity is"normal DNA"polyanionic phosphate backbone.which has negatively chargedIn addition to theTable 2 also reports thereported forhybridization sensitivities,sensitivity for the streptavidin/biotin DNA probe captureaffinity.Table 2 the DNAtransport (accumulation) with low conductivity (B—Alanine,clearly shows correlation ofTaurine, Cysteine, Histidine). The table shows good_2L/20mM NaPO4 adjusted to pH 7.4.W0 98/ 10273101520253035CA 02264780 1999-03-01PCT/US97/ 1448912sensitivity for the streptavidin/biotin. probe affinityreaction using fi—Alanine, Cysteine, and Histidine. Asreflected in the sensitivity data in Table 2, Histidineprovides over four orders of magnitude betterhybridization efficiency then either Cysteine or othersuch as 20 mM NaPO,.Cysteine is at least a factor of 10,factor of 10%10‘. Mosthybridization sensitivitythe Histidine buffer. Thus of allacid buffers presently tested, Histidine is the only oneand good DNA/DNAbuffers, The improvement relative tomore especially aand most especially at least a factor ofthat the DNAis very good forimportantly Table 2 shows(efficiency)the zwitterionic aminowhich provides both good transporthybridization efficiency.It is believed that the low conductivity of thebuffer the rapid DNA(accumulation). There are several possibleHistidine system accounts fortransportexplanations asrelatively efficient DNA/DNA hybridization.may be the good buffering capacity of Histidine.pI at 7.47, Histidine will buffer well under both acidicor basic conditions (see A.L.2ed, Worth Publishers,80). The APEX chip produceselectrode where the DNA is accumulated for hybridization,to why the Histidine buffer producesOne advantageWith itsLehninger, Biochemistry,l975,acid at the positiveNew York, Fig. 4-9 on pageand Histidine may effectively'buffer these conditions.More importantly, under these acidic conditions (pH<5) theprotonation of the imidazole group on the Histidine beginsto convert the molecule into a dicationic species. It maybe the case that this dicationic species with a positivelycharged a-amino group and a positively charge imidazolegroup may help to promote hybridization and stabilize theDNA/DNA hybrids formed at the positive electrode on theAPEX chip. dications, and polycations are knownto help DNA/DNA hybrids by therepulsion of the negatively charged phosphate backbones onthe double—stranded DNA structure.Cations,stabilize reducingIt is also possibleWO 981102731015CA 02264780 1999-03-01PCT/US97/1448913that the DNA/DNA/Histidine may also form some type ofstabilizing adduct from other electrochemical productsbeing produced at the positive electrode (hydrogenperoxide, etc.)While the instant embodiment utilizes naturallyoccurring Histidine, this invention is fully applicable toother natural or synthetic compounds which have good(or zwitterionicwhich allow DNAhybridization to be stabilized by charge stabilization orbuffering capacity, low conductivitycharacteristics) and have propertiesadduct formation.Although the foregoing invention has been describedin some detail by way of illustration and example forpurposes of clarity and understanding, it will be readilyapparent to those of ordinary skill in the art in light ofthe teachings of this invention that certain changes andmodifications may be made thereto without departing fromthe spirit or scope of the appended claims.