CA2079005A1 - Methods and apparatus allowing sequential chemical reactions - Google Patents

Abstract

A first reactant is immobilized i.e. in a porous matrix (50), adjacent a sample electrode (46) within a reaction chamber.
Energizing of the electrode (46) electrophoretically attracts a mobile second reactant and/or electrolytically induces appropriate reaction conditions to enhance reaction of the first and second reactants. Polarity reversals between the sample electrode (46) and remote electrodes (38, 42, 44) cause unreacted second reactant and/or by-products to migrate away from the immobilized first reactant.
The techniques are useful for sequential chemical reactions such as sequencing or construction of proteins, polysaccharides and nucleic acids where cyclical additions and removals of reactants are required. The techniques are amenable to automated micro and nano scale construction and operation and allow direct electrophoretic (38) interfacing with chromatographic, HPCE and mass spectrophotometric equipment.

Description

WO~ 758 PCT/AU91/00141 2 ~ 7 ~

METHODS AND APPARATUS ALLOWING SEQUENTIAL
C~EMICAL REACTIONS
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This invention relates to methods an~ apparatus for carrying out chemical reactions between a plurality of reactants and in particular it is amenable to micro or nano scale operation and to the sequential chemical reactions required during such processes as construction or sequencing of proteins, oligonucleotides and polysaccharides.
A classic example of a sequential chemical reaction is the Edman protein sequencing technique involving the stepwise removal and identification of the N-terminal amino acid residues of a protein. The traditional Edman technique involves coupling the N-terminal amino acid residue to phenylisothiocyanate (PI~C) in a solvent under alkaline or anhydrous conditions to form a . ' ' "'' -' ' . , ' ' ' ,,, : . . ' - : .
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WO91/1575X PCT/~191/00141 2Q7~ 2 ~
phenylthiocarbamyl derivative. Excess PITC (usually at least lO0 fold molar excess) is removed, typically by liquid extraction and the solvent also removed. The N-terminal amino acid, coupled to the PITC is subjected to cleavage by anhydrous acid to form an anilinothiazolinone (ATZ) derivative of the amino acld. The ATZ derivative is removed for subsequent chromatographic identification of the amino acid portion. The original protein is thus truncated, at its N-terminal by one amino acid allowing access to the formerly penultimate amino acid to be coupled, cleaved and identified with a subsequent Edman cycle. Further cycles can be undertaken t~ determine the entire sequence of the proteir..

lS Some of the disadvantages of the traditional liquid phase Edman technique include the necessity to introduce plural incompatible reagents to achieve the coupling and cleavage steps for each cycle. In particular it is noted that strong alkali conditions are required in the coupling step whereas a strong acid environment achieves the cleavage. Clearly differentiation of these strong reagents is required to ensure a consistent removal of all the N-terminal residues without also generating spurious derivatives or uncoupling further non terminal residues. Furthermore the requirement for removal of the various volatile and non volatile auxiliary reagents and solvents during each cycle leads to sample loss and/or the formation of insoluble by-products. Relatively large volumes of sample and reagents are also required.
Effective automation of the Edman technique is first described in Edman and ~egg, "A Protein Sequenator", in the European J. ~iochem. l, (1967), 80-9l and in U.S.
Patent No. 3725010. In these sequenators a liquid phase Edman technique is carried out in a thin film formed on the inside wall of a rotating reaction cell, now termed a "spinning cup". The spinning cup is located within a ' ', ' ...

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WO91/1575~ PCT/AU91/00141 ~ , 3 23~9~
closed reaction chamber to maintain an inert atmosphere.
Reagents are added to the cup by a system of pu~ps and valves and material removed by overflow over the lip of the spinning cup, vacuum evaporation or by dissolving or extractlng in non polar solvents. The liquid reagents and solvents themselves form films on the walls of the spinning cup whlch pass over and interact with the sample film as the cup spins. The rea~ents dissolve the sample film and perform the coupling and cleavage stages of the Edman process, after which volatiles are removed by evacuation and the remaining sample film solvent extracted to transfer resulting amino acid thiazolone for identification.

It will be apparent that this dynamic system, requiring fluid and vacuum seals is dlfficult to construct and operate. The agitation induced by the spinning cup can cause the sample film to be overly extracted during washing or dislodged, in particular if the small proteln being sequenced ls a polypept~de. The initial protein sample must therefore be relatively large in volume and chain length. The drying of the protein onto the lnner wall of the spinning cup must also be performed very carefully and slowly to achieve an even thin film while avoiding boiling and splatter during desiccation. Precise metering of reagents and solvents into the cup is also required to ensure consistency between cycles.

Laursen, in the European J. Biochem. 20 (1971), and Waschter et al., FEBS LeH 35, 97 (1973), described alternative automatic sequencers in which the sample is immobilized by covalent llnkage to the surface of a bead matrix/gel type solid phase. This allows all reagents and solvents to be removed by solvent replacement rather than vacuum evaporation or other drying. Solvent replacement techniques, however, necessitate covalent .

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linkage of the sample to avoid washout by the solvents used. U.S. Patent Nos. 4704256, 4610847 and 4603114 relate to simLlar technologies in wh~ch the sample ls embedded in a permeable matrix and subject to liquid solvents.

Drawbacks of the solvent systems as discussed above have led to gas solid phase Edman techniques, for instance as descrlbed in U.S. Patent Nos. 3892531 and 4065412. In the former patent the sample is attached to a finger like extension withln a reaction chamber, while the latter applies the sample to inner and outer surfaces of macroporous beads. In each system at least one of the reagents is introduced in the gaseous form; however neither system is amenable to contamination free, multiple cycles due to inefficiencies in washing by dew formation on the finger or by channelling of the solvent between rather than through the beads.

Each of the above ~entionQd seyuen~tc_s have required undesirably large samples due to various lnefficlencles ln washing etc. Brltlsh Appllcatlon No.
GB 2146550 describes an attempt to miniaturize a reaction flask suitable for Edman technique having capillary tubes to spray wash interior walls bearing an immobllizing sample matrix. Flulds within the flask can be agitated by the lntroductlon of a gaseous phase. There ls stlll, however, a need for systems more amenable to miniaturization and using even smaller samples. There ls also a need for systems allowing direct interfacing of the chemical reactor with identification or assay apparatus such as mass spectrometers, chromatographs etc.

The above descrlbed Edman technique for terminal degradation of a protein, successively removed N-terminal residues. Alternative systems using a C-terminal degradation, such as phosphoryl mercaptobenzothiazole or ' ~

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WO91/t~758 PCT/AU91/00141 2~ 7~
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benzoyl isothiocyanate are described in "Methods in Protein Sequence Analysis" (1991) Jornvall/H~og/-Gustavsson (Eds) ~irkhauser Verlag, Basel. The latter system embodies a two stage couple/cleave reaction between the cleaving reagent and C-terminal amino acid, rather like the Edman technlque. Further guidance on protein sequenclng is founding i~ Schlack et al. ~1926) Hoppe-Seylers, Z. Physiol. Chemie., 154, 126-170 and-Stark (1972) "Methods in Enzymology", Hirs~Timascheff (Eds) Academlc Press, 25, 369.

The above description of previous techniques and apparatus has centred on protein sequencing, but it will be readily apparent that many other chemical processes face similar problems of sample loss or contamination, inefficient or overly vigorous washing, inefficient access of reagents etc. Examples of such reactions includes site specific modification, construction or sequencing of other macromolecules.
In accordance with a first aspect of the present invention there is provided a method of conductlng a chemical reaction, the method comprising immobilizing a first reactant at a reaction locus, electrophoretically introducing to the reaction locus at least one charged, addltional reactant and after reaction between the flrst and addltional reactants removing the remaining additional reactant and/or a reaction by-product of the reaction from the reaction locus.
A related aspect of the invention provides chemical reaction apparatus, comprising a reaction locus, immobilizing means for securing a first reactant at the reaction locus, means for the electrophoretic introduction to the reaction locus of at least one additional reactant and means to remove, from the locus, the remaining additional reactant and/or a reaction by--: . - .
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WO91~lS758 PCT/A~I9l/001~1 2 ~ 6 ~`
product of the first and additional reactants.

By the use of the present invention the electrophoretic introductlon of the additional reactant causes it to focus or concentrate around the electrode upon whlch is immobillzed the flrst reagent. Thus a relative~y dilute concentration of the additional reactant can achieve enhanced concentrations and therefore reaction efficiencies. Comparatively weak feed solutions of the additional reactant have the advantage of facilitating washing out thereof prior to subsequent reactant additions, thereby minimizing contaminating carry-over.

ln the context of this specification "chemical reactions" is being used in its classical sense denoting a molecular level interaction between the first and additional reactants resulting in a change in structure of one or both reactants and includes such interactions as enzymati~ reactions Furtherm^.e, "el2ct-cpho,e~ic"
includes "electrokinetic".

The first reactant will typically be immobilized immediately adjacent an electrode serving tO cause electrophoretic migration of the charged additional reactant. Preferably the first reactant is immobilized on such an electrode itself or on a sample matrix extension thereof.

At its simplest, the above mentioned means to remove remaining additional reactant and/or reactant by-product flush these materials away from the reaction locus.
Suitably controlled syringes or pumps can be used to achieve this. The method and apparatus of this aspect of the invention may however be adapted to the collection and/or identification of a reaction by-product of the first and an additional reactant. A convenient ., : , ' : , . ~ . , .
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arrangement where the by-product is itself charged or can be made charged provides electrophoretic or electrokinetic means to selectively remove the by-product for subsequent analysis away from the reaction locus by, for instance, mass spectrometry or capillary electrophoresis. These analytlcal techniq~les use extremely small samples. Rs the apparatus and method of the invention are amenable to micro and nano scale reactions, direct injection of the by-product described above into the sample port of the analysis instrument becomes possible thus minimizing losses and inaccuracies which may occur, for instance, during sample dilution in prior art methods. Other appropriate analysis equipment may involve electromagnetic absorption, fluorescence, nuclear resonance, immunological detection, gas liquid chromatography GLC etc.

The above mentioned arrangement having electrophoretic removal of reaction by-products ls not necessarily limited to a set-up ln which the additional reactant~s) is/are introduced electrophoretically.

Accordingly, a second aspect of the invention provides a method of conducting a chemical reaction, the method comprising immobilizing a first reactant at a reaction locus, introducing an additional reactant to the reaction locus, allowing a reaction between the first and additional reactants to produce a reaction by-product, if the reaction by-product is not charged, imparting an electrical charge to the reaction by-product and electrophoretically removing, from the reaction locus, the by-product for collection or analysis. Conveniently this electrophoretic removal directly injects the by-product into an analysis instrument as described immediately above.

A related aspect of the invention provides apparatus ' ' : . . .
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WO91/1~75~ PCT/A~!91/001~1 2~790~ 8 ~
for conductlng a chemical reaction, the apparatus comprising a reaction locus, immobilizing means for securing a first reactant at the reaction locus, means to introduce an additional reactant to the reaction locus, optionally means to impart a charge to a reaction by-product of a reaction between the first and add{tional by-products and means to electrophoretically remove from the reaction locus, the reaction by-product.

Conveniently an ion exchange membrane intermediate the reaction locus and the injection port is used to separate the by-product from other species in the reaction portio~ of the apparatus. Ion exchange membranes such as BIO-REX products having AG ion exchange lS resin beads permanently enmeshed in a polyfluoroethylene matrix are available with a number of ion exchange varieties suitable for separating different species of by-products, both cationic and anionic.

Where a reaction by-product is directly in~ected into the sampIe port of an analysis apparatus, electrophoretically or otherwise, it is desirable to employ a phase contrast between the reaction locus medium and the analysis medium. For instance where the medium around the reaction locus comprises a non polar medium, a polar medium in the sample injection port of the analysis apparatus in con~unction with a suitable hydrophobic membrane can assist in separating the by-product from the reaction solvent or diluent. An example can be taken from the above noted Edman technique which utillses an organic medium. In contrast a capillary electrophoresis analyser can run on an aqueous medium. Separation of the by-product is efflciently achieved through a hydrophobic reaction locus medium by a porous hydrophobic membrane composed of polytetrafluoroethylene. Conversely in other reaction systems an aqueous reaction medium around the reaction locus may encourage the use of non polar : . , . -' ' , . ! ~ , . ~ : , - , , ,: : . . :
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' ' . , ' ' , ~ . ' WO91/l57~ PCT/Al'91/00141 `' 9 chromatographic detection techniques in conjunction wlth porous hydrophilic membranes.

The methods and apparatus defined above may be particularly suited to sequential chemical reactions between the first reactant and a plurality of lndivldual second and subsequent reactants. Thus a first reaction may be commenced by electrophoretically introducing a second reactant to the first reactant followins which unreacted second reactant and reaction by-products can be flushed away or electrophoretically removed. Thereafter a third reactant may be (electrophoretically) introduced to the reaction locus for reaction with an immobilized reaction product of the first and second-reactants. In this way an ordered sequence of reaction products, as may be used in the construction of macromolecules such as polysaccharides, oligonucleotides or proteins may be built up on the immobilized first reactant. It will of course be necessary to provide such additional reactant "building blocks" with a suitable charae to ensure thoi-electrophoretic migration to the "foundation" first reactant. It will also be necessary, if a macromolecule having an ordered sequence is to be built up, to ensure that the additional reactant bonds at the desired terminal of the incipient macromolecule. To this end appropriate capping enzymes (such as glycosyl transferases for polysaccharides or ribosomal transferases for proteins) can be introduced lnto the reaction medium. Alternatively acid or base catalysed condensations can be enhanced by in sl tu acid/base generation as described later.

Alternative sequential chemical reactions occur in protein, oligonucleotide or polysaccharide sequencing.
~he prior art discussion above in relation to the Edman technique or C-terminal degradation of a protein is an example of such sequencing reactions. When the present ; PCT/A~191/00141 l v ~ ~

invention is applled to such sequencing reactions, the first reactant can comprise the protein, oligonucleotide or polysaccharide to be sequenced, while the addltlonal reactant may comprise a cleaving agent, successive aliquots of which can be lntroduced to the macromolecule first reactant. The cleaved off by-product i.e. amino acid, nucleiç acld or saccharide, can be collected for analysis after the reaction ~f the macromolecule with each aliquot of cleaving agent and before the next aliquot of cleaving agent is introduced to the immobilized macromolecule. ~y appropriate choice of charge for the respective reactants and ~y-products, the additional reactant and/or by-product of the reaction can be electrophoretically separated for subsequent analysis of the by-product and/or collection of unreacted additional reactant for reuse or discard.

Furthermore, if unreacted additional reactant is electrophoretically removed from the reaction locus and collected at a collestion 10~UQ -d,--e-,' 2 colleot'o., electrode lt is possible, by reversing the respective voltages of collection and reaction locus electrodes, to cause a second aliquot of the additional reactant for instance a cleaving reagent in a sequencing system to be reintroduced to the reaction locus from the collection locus. The collection locus thus operates as a reservoir for the additional reactant.

Alternative reactions involving macromolecules and amenable to the present invention include the immobilizing of an enzyme such as a restriction endonuclease or endopeptidase to allow analysis of the nucleotide or polypeptide structure not requiring end terminal truncation. Conversely a peptide or nucleic acid of interest could be immobilized and subsequently introduced to a series of peptidase or nucleases to determine cleavage pattern. Such an approach is useful '~ . ' - ' . :

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W~91/1~75~ PCT/AU91/00141 11 2~7~
ln determining, eg. if fusion proteins have been produced by a recombinant host or in DNA restriction analysis.

It is also contemplated that the structure of macromolecules other than proteins, polysaccharides and nucleotides such as long chain lipids, glycoproteins and glycolipids c3uld be determined using techniques of the invention, typically utilizin~ enzymic clea~age.

A useful feature forming a third aspect of the invention but also observed in the above described electrophoretic apparatus and methods is that energizing of an electrode having the first reactant attached thereto can electroactivate reagents in the medium nearby. For instance a negatively charged electrode will attract positively charged reactants. Additionally it can electrolyse, to some extent, reagents or solvents nearby or in some circumstances the first reactant. Such electrolysis can assist in activation of certain reactions or provide acid or base conditions im~.ediate1, adJacent the electrode. For instance in the earlier discussed classic Edman technique, a base environment is required to react the Edman reagent i.e. the lsothiocyanate couplins agent to the N-terminal amino acid. Traditionally the base conditions are provided by adding a strong base such as Quadrol (Wyandotte Chemicals Corp.) buffer to the reaction vessel. It has now been discovered, however, that baslc conditions can be generated i~ situ adjacent the sample by electrolysis following energizing of the electrode bearing the first reagent. Similarly acid conditions required, for instance, in the cleavage step of Edman procedures can be generated in situ by positive charge energizing of the electrode. It should be noted that this aspect of the invention is not limited to electrophoretically transferred reagents as non charged Edman reagents such as PITC can also be "electroactivated" by basic WO91/1575~ PCT/A~91/00141 2~ 3 12 ~
condltlons produced by a negatlvely charged electrode.

Accordingly a third aspect of the inventlon provldes a method of conducting a chemical reaction, the method comprising immobilizing a first reactant at an electrode defining a reaction locus, introducin~ at least one additional reactant to the reaction locus, electrolytically activating a reaction between the first and additional reactants by energizing the electrode and thereafter removing from the reaction locus, remalning additional reactant and/or a reaction by-product of the reaction.

Further provided by this invention and related to lS this third aspect, is che~mlcal reaction apparatus comprising an electrode defining a reaction locus, means to immobilize a first reactant at the reaction locus, means to introduce an additional reactant to the reaction locus, means to energize the electrode thereby electrolytically actlvating a reaction between .he flrst and addltional reactants and means to remove from the reactlon locus remalning additlonal reactant and/or a reaction by-product of the reaction.
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With this aspect of the invention, the electroactivation of the reaction between first and additional reactants can either be by in situ generation of appropriate pH conditions to enhance reaction ~lnetlcs (as with the Edman technique example described immediately above). It will also be apparent that ln alternative reaction systems active radicals can be formed in situ, adjacent the electrodes by direct electrolysis of the first or more usually the additional reactant.
Electrolysis to product electroactivated species will occur most efficiently where the field strength is : ' : . ., ' . :. . : : :
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WO91/1~75R PCT/AU~1/00141 ~``"` 13 2 ~ 7 ~
strongest. It is thus deslrable to immobilize the flrst reactant immediately ad~acent a conductor such as a platinum, such that the electric field strength through the first reactant is maxlmlzed. In effect a sample matrlx lmmobillzing the first reactant becomes an extension of the electrode itself.

Referring still to the "electroactivating" aspect of the invention, as with the earlier described first aspect of the invention, the means to remove the remainlng additional reactant and/or reaction by-product may simply comprise a pump or syringe to flush the reaction locus.
Preferably however, the method and apparatus allow the collection and/or identification of a reaction by-product. As described above electrophoretic means can beprovided to selectively remove the by-product, if lt ls charged, for subsequent analysis away from the reaction locus. Where a by-product does not inherently bear a charge, introduction of charged surfactants, such as SDS
sodium dodecyl sulphate can render such materials electrophoretically migratable.

It will be apparent that each of the above described aspects of the invention are amenable to micro and nano scale construction and operation. Reaction locus volumes of less than l x 106 nl, preferably l x 103 nl and even of the order of lO nl to lO0 nl are achievable and desirable. Correspondingly small ampages of electrolytlc or electrophoretic current, of the order of fractions of a milliamp - for instance .Ol to lO ~A can be used. Low currents assist in preventing temperature surges in the cell. This has many advantages over current more macro scale techniques in macromolecular construction and sequencing. It allows the use of extremely small amounts of the reactant immobilized at the reaction locus. This is particularly important in sequencing work where the nucleotide, protein or polysaccharide may not be . ,.,.. ~ . . ~ . .
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WO91/15758 PCT/A~'91/00141 ,~79i~ 14 ~
avallable in large quantltles or, as has been discovered during the use of prior art techniques discussed above, may be difficult to manipulate in small quantities. As mentloned above a micro or nano scale operation allows direct injection of reaction by-products (i.e. cleavage residues) into highly sensitive analysis apparatus wlthout requiring dilution or other preparation steps.

Referring still to the advantages of micro or nano scale operation, dlfficultles ln effectlvely washing the reaction locus without disturbing the immobilized reagent, a particular problem in many prior art -techniques, is lessened in the present invention where comparatively small amounts of reagents, diluents and washes can be used. ~n this regard it will be recalled that in the electrophoretic aspect of the invention, comparatlve weak reagent concentrations, can be tolerated due to the electrophoretic focussing of the active ~-reagent at the reaction slte. Weakly conce~trated reagents are of course more easily removed and washed away than more concentrated solutlons known in the prior art. Additionally, or alternatively, in the electrolytic aspect of the invention, electroactivation in situ of reagents can allow the use of less concentrated reagent feedstock, and/or eliminate the need to add strong pH
control agents. Again the effect of this is to facilltate removal of unwanted reagents and by-products thus preventing carry-over and contamination of subsequent reactions at the reaction locus.
With any of the above defined aspects of the invention, immobilization of the first reactant, typically on an electrode is required. Several of the prior art techniques discussed above have immobilized ~5 protelns via covalent linkages. Securing the reagent strongly was important to prevent loss of the sample during the various reactions and washings but is , ... . , - . . ., . - . .~:
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- . , W091~1~ 5~ PCT/AU91/00141 dlfficult to achieve without adversely affecting the reagent. In contrast the present invention allows gentler methods of attachment of the first reagent at the reaction locus. Desirably the first reagent can be adsorbed directly onto a porous matrix which allows access of the additional reagents to the first reactant particularly its end term$nals. Many such matr.ices are known for polysaccharides, proteins and nucleotides.
Examples of such matrices include PVDF, nitrocellulose, glass, quartz, activated glass, activated quartz, cellulose, derivatized cellulose, nylon, plastic or activated plastic. Adsorption in this fashion, unlike covalent binding assists in preserving the structure of the first reagent which is particularly important in sequencing_and macromolecule construction operations.

Reaction kinetics will, of course, differ between different combinations of reactants. In the case of a sequential chemical reaction, such as terminal degradation sequencing each res~due alons the chaLr~ ay have differing kinetics of reaction wlth a cleavage reagent. Proline, for instance, in Edman degradation techniques requires a longer reaction time to achieve complete coupling than other amino acids. It is thus desirable to be able to monitor extent of reaction within the cell. This can be achieved colorimetrically by utilizing redox indicators which change colour or absorbance as the redox potential at the reaction locus changes. Other spectrophotometric techniques including laser induced fluorescence are also available which have the advantage of not requiring additional reagents at the reaction locus. Alternatively the extent of reaction can be measured amperometrically by monitoring current through an electrode defining the reaction locus. Thus a plateauing of electrolytic activation can be used to indicate completion of a coupling or cleavage reagent.

.

WO91~1~75~ PCT/A~'91/00141 2 ~ 7 ~
Varlous embodlments of apparatus and methods in accordance with the invention will now be described by way of example only with reference to the following Examples and to the accompanying drawings in whlch:
Fig. l is a schematic plan of a first embodlment of the reactlon apparatus in con~unction with ancillary analysis and control equ$pment;
Fig. 2 is a sectional side view of the reac~ion apparatus of Fig. l;
Fig. 3 is a schematic plan view of a second embodiment of reaction apparatus in accordance wlth the lnve~tion;
Fig. 4 is a partial sectional side view of the embodlment of Fig. 3;
Fig. 5 is a graph depicting amino acid determination data of experiments A to D in Example l; and Fig. 6 is a graph depicting amino acld determination data of experlments A to E ln Example 2.

These embodiments of arpar2~us and m2 ~hods -will be described with reference to proteln sequenclng by the Edman sequentlal degradation process. In short, this appllcation of the invention involves immobilizing a protein sample on an electrode at a reaction locus, for instance, withln a glass ele~trophoresis chamber. A
first aliquot of a cleaving agent is caused to electrophoretically mlgrate to the reactlon locus to cleave an end amlno acld from the immobillzed protein.
The cleaved amino acid molecules are then electrophoretlcally transferred to an analysis apparatus, in this case hlgh performance capillary electrophoresls apparatus for identification thereof. Subsequently, second and further aliquots of the cleaving agent can be electrophoretically introduced to the reaction locus to sequentially cleave the remaining amino acids and thus allow the sequence to be determined. The achievement of this method will be discussed in further detail below.

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Referring initially to Fig. 1, the reaction apparatus lO is depicted in conjunction with ancillary computer control deYice 12 and amino acid analysis apparatus 14 in the form of a capillary zone electrophoresis (HPCE) apparatus of known construction (see for instance Gordon, M.J. et al. (1988) "Capillary Electrophoresis Science", ~242):242-248).

In short, the HPCE performs electrophoresis of very small ~uantities (5-50 nL) within capillary tubing typically of slOO um i.d. In the schematically depicted apparatus 14, the electrophoretic separation column 16 has its sample injection por. 18 opening directly into a reaction chamber 20 of the reaction apparatus lO. This arrangement is described in detail below with reference to Fig. 2. The remaining major functions of the HPCE are a detector 22 monitoring the separated species from the column 16, and a hlgh voltage source 24 in communicatlon with the in~ection port 18 and column 16 through buffer line 26.

The reaction apparatus 10, in addition to the above mentioned reaction chamber 20 includes a plurality of cleavage reactant and flushing agent syringes 28 leading into the chamber 20 ~ia inflow line 30. Leading from the chamber 20 is outflow 32 to waste.

A plurallty of electrodes (not deplcted in figure 1) each wlth a respective lead, are disposed within the reaction chamber. Each electrode is independently under the control of the computer 12, as are the syringes 28 enabling the method of the invention to be automated.
The computer 12 further receives data from the detector 22 of the HPCE 14.
Turning now to Fig. 2, the reaction apparatus 10 comprises the electrophoretic reaction cham~er 20 defined ~O91/lS/~8 PCT/A~'91/00141 2 ~ 7 ~ 18 ~
between a plurality of closely fitted and secured glass blocks 34. Clearly, other inert materials such as glass, quartz, ceramic, plastics etc. may be employed for the construction of a suitable chamber within which to conduct the electrophoretic transfers required by the invention. Conveniently, at least a portion of the material defining the chamber is transparent to allow the use of optical monitoring e~uipment such as an absorbance spectrophotometer to gauge the progress of reactions at the reaction locus and/or monitor the transfer of reactants or by-products to or from the locus.
Advantageously, the material defining the chamber allows the temperature of the contents c r the chamber to be kep.
steady. This may be achieved by an active heat transfer system ~uch as a peltier device.

As mentioned above, leading into the reaction chamber 20, but separated therefrom by a hydrophobic porous PTFE membrane 36 is the sample injection port 18, buffer line 26 and capillary column 16 of the HPCE. ~-electrode 38 is mounted within the injectlon port. For simplicity, the electric lead 38' connecting the electrode 38 to the voltage source/computer 12 has been truncated.
Three further electrodes, 40, 42 and 44 are disposed on an interior wall of the reaction chamber 20 successlvely downstream of a sample electrode 46 dlsposed facing the membrane 36 over the HPCE injection port.
Each of the electrodes 40, 42, 44 and 46 has a respective lead 40', 42', 44' or 46' which have also been truncated for clarity but lead to the computer 12 for adjustment of respective voltages applied between the various electrodes. Electrode 44, most remote from the sample electrode 46 is disposed on an interior wall portion of an expanded portion 48 of the reaction chamber defining an electrophoretic storage locus as described further , : . . : : . , ' ' . : : ' - . .: ~ : , . . . ..
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below.

When fitted, the sample electrode 46 terminates, within the reaction chamber, at a chemically inert protein immobilizing matrix 50 such as a polyvlnylidene difluoride porous membrane. The sample electrode 46 further comprises a removable threaded support 52 to disengageably secure the electrode through the chamber walls. The sample eleotrode in this embodiment is platinum but other electrically conductive materials such as gold, tungsten or graphite may also be appropriate.

The apparatus is adapted to miniaturization enabling fine temperature and environmental control thereby to optlmize the sequential degradation process. The reaction chamber may be, for instance, of the order of a millimetre or significantly less across allowing the use of very low (~A) currents for the electrophoretic transfers. This further assists in preventing temperature surges whi-h may effec thG che.mical reaction.

Sam~le loadin~
In use, the sample (for example, protein or oligopeptide) is immobilized by application, external to the apparatus, to the chemically inert immobilizing matrix (e.g. porous polyvinylidene difluoride (PVDF) membrane 50) afflxed to the end of the removable, insulated (PTFE) platinum electrode 46 either by passive means (e.g. direct spotting) or electrotransfer. After sample immobilization, sample electrode 46 is re-installed into the glass blocks defining the reaction chamber so that the end of electrode 46 with the membrane 50 and the immobilized sample is positioned on the internal surface of the electrophoretic reaction chamber 20.

WO91/157~Y PCT/~191/00141 2 ~7 9 ~i3 ~ 20 ~ he remalnder of the sequencing operation ls cycllc and includes the following steps:

l. Initial reaction Isothiocyanate coupling of immobilized protein or peptlde amino groups is achieved by electrotransfer of a positively-charged lsothiocyanate te.g. rhodamine ~) dissolved ln an anhydrous organic solvent (e.g. lO~ n-propanol/90~ butyl chloride); to the negative sample electrode 46, the basic conditions required for efficient coupling of isothiocyanates to non-protonated amine groups on the proteins or peptides immobilized at electrode 46 are achieved by the use of an organic solvent soluble acid-base salt (e.g.
triethylammonium trifluoroacetate) wherein decomposition to a basic component occurs at the negative sample electrode. The positive electrode in this electrical field ~Field l) ls electrode 44. Electrophoretic migration of the pigmented and fluorescent rhodamlne ITC
can be monitored visuzl~y through -he t,a,-sparent glass walls of the cells as can focussing and concentration of the reagent ad~acent the electrode 46.

2. Removal of excess reaoent After coupling, unreacted rhodamine ~ isothiocyanate and lts by-products (e.g. thioureas) are electro-phoretically transferred to the storage locus 48 at the expanded portion of the reactlon chamber using electrode 44 by reversing the polarity of electrodes 46 and 44 (i.e. electrode 46 is now positive with respect to electrode 44).

As in all Edman degradation techniques it is important to achieve a lO0~ cleavage of each terminal amino acid residue in each cycle. If this is not achieved, the next cycle of coupling and cleavage may be contaminated with terminal amino acids left uncleaved .
' . ~ ' , . - ~

W091/1~75~ PCT/AU91/00141 ~ 21 2~
from the previous cycle. In the illustrated embodiment employing HPCE detection of cleaved residues, extremely small numbers of residues are adequate to achieve an unambiguous identification. Accordingly a relatlvely small coupling efficiency of the charged ITC reagent can be tolerated. If such a low efficiency coupling reagent ls used, lt ls advisable to introduce a high efficiency Edman reagent after coupling of the electrophoretic moiety to couple to and fill any remaining N-terminal residues thereby to ensure lO0~ cleavage. Accordingly lt is desirable to flush the reaction cell with a phenyl-ITC
(PITC) reagent of high coupling efficiency after coupling of the rhodamine I~C. As PITC does not bear an electric charge it cannot be electrophoresed and thus is injected by syrlnge; Removal of unreacted PITC requires flushlng of the cell. It is desirable to accomplish thls prior to cleavage of reacted ITC derivatives to ensure that penultlmate N-terminal resldues are not prematurely coupled by PITC although the baslc conditions required for such coupling will g~ner2lly quickly d~ssipatO ~her.
electrolysis around the electrode is ceased.

3. Cleavaae ste~
Concomitant with the polarity reversal of electrodes 46 and 44, decomposltlon occurs at the posltlvely-charged sample electrode 46 as cleavage of the rhodamlne B
thiocarbamyl amino acid from the immobilized protein or peptide commences. Since this cleavage reaction is relatively slow compared to the electrophoretic movement of unreacted rhodamine B isothiocyanate and its by-products (e.g. thiourea), these latter species will pass electrode 40 before the cleavage reaction has proceeded to any appreciable extent.

4. Transfer of isothiocYanate derivative Electrode 40 is now made positive with respect to electrode 44, creating an electric field (Field 2) and WO91/15758 PCT/A~'91/00141 ?~ 9~ 22 the electrophoretic mlgration of the rhodamlne B
isothiocyanate products continues towards electrode 44.
When these products pass electrode 42, this electrode is made positive with respect to electrode 44 (but not as posltive as electrode 40) to create an electric field ~Field 3) which entraps rhodamlne B isothiocyanate near electrode 44 whilst the sample electrode 46 is totally lsolated, electrlcally, from electrodes 44, 42 and 40.
At this stage the polarity of electrode 46 is made positive with respect to electrode 38 situated in ~he injection por~ 18 of the "capillary electrophoresis"
component of the apparatus. The electric field (Field 4) created allows further accumulation of acid and cleavage to occur at the sample electrode 46; the cleaved thiazolinone amino acld derlvative (positively charged) migrate through the porous membrane 36 towards electrode 38 in the HPCE sample injection port 18. Separation of the capillary electrophoresis aqueous buffer within the capillary from the anhydrous or~anic solvent in the reaction chamber is achieved by the hydrnphoblc m~brane 36.

S. Identification of thiazolinone amino acid derivatives After the completion of the cleavage reaction, the thiazolinone amino acid derivatives which have now accumulated at electrode 38 are identified and quantified with the "capillary electrophoresis" system (for this purpose, electrode 38 is isolated). To further enhance selectivity and sensitivity of detection, the hlgh separation efficiencies of capillary zone electrophoresls can be combined with sensitive fluorescence or mass spectrometric detection (for details said Smith, R.D. et al. (1988) "Capillary Zone Electrophoresis - Mass Spectrometry", Anal. Chem. 60:436-441).

' : ' - ' , ' ~ ' .. . . ~: , . :
, WO91/I~,SX PCT/A~'91/001~1 23 2 ~ 7~-J!5 6. Subse~uent cleavaae cYcles The above-mentioned cycle of the Edman degradatlon procedure comprising steps 1-5 can be repeated by making the polarity of sample electrode, 46, negative with respect to electrode 44. Alternatively, the reagents and solvent in electrophoretic reaction chamber may be replenished during or at the end of each cycle via the syringes 28. If required, additional reagents for coupling, cleavage or flushing solvents may be introduced into the chamber 20 by the use of one or more of the syringes 28 connected to the input 30.

In the above described embodimen., the positively charged rhodamine derivative of ITC was used and this moiety therefore electrophoretically migrated to the reaction locus. The use of such a positively charged reagent is particularly advantageous in a system such as Edman degradation where the coupling reaction requires basic conditions as in situ generation of the base was also occurring at the reaction locus as an a-~xi li2r~ sal' within the medium, triethylammonium trifluoracetate was being electrolysed.

In an alternative acid catalysed system, a negatively charged group such as fluorescein could be attached to a cleavage reagent and electrophoretically attracted towards a positively charged electrode. At the positive electrode electrolysis of an auxiliary reagent or the medium itself could, simultaneously, be creating acid co~ditions adjacent the first reagent. Clearly where the reaction between first and additional reagents does not required a pH shift, the medium and any auxiliary reagents would be selected to ensure minimal electrolytic action adjacent the electrode. Use of a fluorescent charged group is convenient as it allows easy optical monitoring of reactant transfer and can also assist in combined fluorescent/CE determinations.

WO 91/157~8 PCT/A~'91/00141 2 ~ 7 Q ~ ~ ~ Referring now to Figs. 3 and 4 an alternative but generally similar microcell is depicted. In this embodiment the microcell 100, of milled borosilica glass comprises a lower portion 102 having etched therein a longltudinal groove 104 intersected by upstream 106 and downstream 108 sets of three parallel transverse grooves.
Bored through the lower portion 102 intermediate the sets 106, 108 of grooves is a vertically extending recess 110 into which is screwed, in use, a 0.8 mm diameter noble me~al sample electrode 112 as best seen in Fig. 4.
Extending around the electrode is an insulating sheath 114 of PTFE. Sealingly extending over the lower portion 102 is a glass upper portion 116 defining the roof of recess llO and grooves 104, 106 and 108. The glass upper portion allows direct microscopic observation of the interior of the apparatus and hence allows colorimetric ind$cation of the transfer of reactants. The g7ass upper cover is omitted in Fig. 3.

In this embodiment recess 110 dsfine~ the rsac_i 3n chamber. Longitudinal groove 104 extending into the chamber defines 0.09 mm diameter upstream 118 and downstream 120 conduit for reagent entry and removal.
Extending along each of the transverse grooves in direct electrical communication with the adjacent intersecting conduit 118 or 120 is a respective gold electrode (0.08 mm).

Referring now to Fig. 4, it can be seen that a 0.15 mm deep sample matrix 122 overlies the sample electrode 112. The insulating sheath 114 extends upwardly at reference numeral 124 around the sample matrix although a gap 126 between the sheath and the glass top portion 116 allows entry and exit of reactants from the conduits to the sample matrix. In the described arrangement, the sample matrix 122 of PVDF which except for the insulating sheath, fills recess 110 has a nominal .
. - .

, :

0 91/1~-58 PCT/A~'91/00141 2~7~
volume 75 nl but wlth an 80% void volume. The effective reaction chamber ln this embodiment ls thus 60 nl plus respectlve volumes of about l nl on either side between the recess llO and the nearest electrode upstream and downstream (respectively 128, 130).

Not depicted in Figs. 3 and 4 are the downstream ~analytical device, upstream reagent injection apparatus at manifold 128 - Kloehn model 50100 syringe drives, lO0 ~l syringes, electrical connections between electrodes 112, 118 and 120, power source and microprocessor controller. It is noted however that the spaced electrodes are each independently variable in voltage and polarity. The respective upstream and downstream electrical sets 118 and 120 thus allow for a focusing and electrophoretic movemeht of charged reagents, somewhat analogous to a peristaltic wave. They can also serve as holding reservoirs as discussed with regard to the embodiment of Flgs. l and 2.
Also not indicated in Figs. 3 and 4 is the amperometric apparatus which measures current flow through the sample electrode 112. Completion of a reaction in the cell is indicated by a plateauing of the current flow in the electrode. This amperometric determination allows easy interface of the cell with a microprocessor controlling the syringe drives responsible for introducing new reactants. The timing of the various steps within a cycle of a sequential step can thus be set according to the reaction kinetics in the particular combination of reactants in the cell.

1. APPARATUS

A simplified version of the apparatus shown in Fig.
2, but lacking the capillary electrophoresis interface, was utilized in this Example. The platinum electrode (46 WO 91tl~75~ PCr/A~191/0014i 2~7~`~1`3~ 26 in Fig. 2) with an affixed PVDF membrane for sample application (50 in Fig. 2) was 5 mm in diameter. One layer of cellulose paper (Whatman 3MM, 5 mm diam.) was positioned over the PVDF to minimize diffusion of electrolytlcally generated reagents in the cell. The electrode was insulated (PTFE) so that electric current from the electrode passed through the PVDF. The electrode was positioned in the cell (4-ml glass vessel wlth,a loose-fitting cap), together wlth a second simple platinum,electrode wire. The electrodes were positioned 8 mm apart and connected to a constant power supply.

2. SAMPLE LOADING

The sample (200 picomoles ~-lactoglobu1in) was immobilized to the PVDF membrane (50), affixed to the sample platinum electrode (46) by passive means.
~-lactoglobulin is a 18-kDa protein with the following N-termlnal sequence: H2N-Leu-Ile-Val-Thr- (for details see Braunltzerr G. et al. (lq72) 7 . Physiol. Chem~e., 353:832-834).

3. EDMAN DEGRADATION

(a) COUPLING The cell was filled with 3.5 ml of butylchloride containlng 5~ (v/v) phenyl isothlocyanate, 5~ (v/v) triethyl ammonium trifluoroacetate and 0.2~
(w/v) dithiothreitol. The cell was flushed with argon, heated to 50C (air oven) and connected to a constant 30 power supply (200 ~A, 20-30V) for 60 mins with the sample electrode (46) negative. The sample electrode was then removed from the cell, briefly washed ( 30 sec) wlth 0.5 ml ethylacetate and returned to the cell for the cleavage reaction.
(b) CLEAVAGE The cell was filled with 3.5 ml butylchloride containing 10~ (v/v) trifluoroacetic acid ; -- , , . . ~ -: . ' ' ' ~ ' ' ' ' ,. .

.. , : . . .
. .

WO91/1~75~ PCT/AU9t/00141 ~ 27 2~7~
and 0.2% (w/v) dithiothreitol, heated to 50~C (air oven) and connected (sample electrode, positive) to the power supply (36V, 50,uA) for 30 mins. The sample electrode was then removed from the cell.

4. DETERMINATION OF REMAlNING N-TERMINAL RESIDUES

The PVDF membrane was removed from the sample electrode, washed with 0.5 ml ethyl acetate, and placed in the sample cartridge of an Applied Biosystems Sequencer (model 470A) equipped with an on-line phenylthiohydantoin (PTH) - amino acid analyzer.
Sequence analysis of the N-terminal residues of the ~-lactoglobulin remaining on the membrane was performed using regular Applied Biosystems cycles and program 03RTST. The sequencing results are shown in Fig. 5.

Legend to Fig. 5:
A Control (~-lactoglobulin sequence was determined directlv in 470A i.e. omitting step 3) B One Edman degradation cycle was performed including electrolytic generation of the basic and acidic conditions required for coupling and cleavage and the remalning N-terminal residues sequenced in 470A

C Control for 'B' (i.e., identical treatment as in B, but no electrolysis) D Two Edman degradation cycles were performed electrolytically and the remaining N-terminal residues sequenced in 470A

' ~ ' ' ' ' ' . . .' .. ' ' - , . .- . . : - . . ~ .

W091/1575~ PCT/A~'91/00141 2 ~ 7 ~ 8 Note: -(1) One letter notation used for amino acids i.e., L, leucine; I, lsoleucine and V, valine.
(2) Cycle 1, 2 and 3 in the leftmost legend corresponds to the first, second and third cycles of the 470A sequenclng of the N termlnal residues remaining on the respective membrane.

5. COMMENTS

(a) In Ex~t B; The presence of Ile ln cycle 1, Val in cycle 2 (Thr in cycle 3 was too low to be detected) is clear evidence that the N-terminal leucine has been removed by the electrolytic coupling and cleavage procedure described in 3(a) and 3(b).

(b) confirmation that the removal of N-terminal leucine was due to the electrolytic process and not Just due to passive exposure to the reagents is provlded by the data in ExDt C i.e., Compare Ile levels in cycle 1 (Val levels in cycle 2) in Expts B and C. In Ex~t C, ~-lactoglobulin was exposed to the same regimen of chemicals as in B for the same time and temperatùre but no electrolysis was performed.

(c) In Ex~t D: The presence of increased levels of Val in cycle 1 is indicative of two successful cycles of Edman degradation having been performed by the electrolytic procedure described in 3(a) and 3(b) (i.e., compare Val level in Expt D with those in B and C - cycle 1) ~

(d) In the above described experiments NO attempt has been made to optimize the kinetics/chemistry of the coupllng/cleavage processes.

- . ': : . . - -,,.
.
. ... . . : , . , . :~
.

29 2~7~

This Example is similar in intent to Example l but performed on the micro/nano scale appara~us described by reference to Figs. 3 and 4. The Example included the following experiments:

(A) Control SummarY
- ~-lactoglobulin is adsorbed to sample matrix and then sequenced using a standard ABI 420 Sequencer.
Procedure - ~-lactoglobulin adsorbed onto PVDF disc 0.8 mm diameter O.15 mm thick. PVDF discs (= 50) prewetted in acetonitrile then lmmersed in l ml 10%
acetonitrile lO mgm ~-lactoglobulir. fo- l hour at 20C. Protein solution poured off the discs, washed l0 times ~z 5 ml) 10% acetonitrile. Discs washed l0 times ln water then dried in freeze dryer stored under argon at -20C. One disc placed into ABI 470 Sequencer with preconditioned polybrene glass disc.
Normal PTH cycles.

(B) ~a~Dl e of El ectrol vtic CouD~nq Summar~
- ~-lactoglobulin is adsorbed to sample matrix and the matrix is disposed in the microcell of Figs. 3 and 4. PITC Edman reagent is introduced to the microcell where basic conditions are electrolytically generated in situ. The sample matrix is then sequenced with ABI 470.

. . ::: . : '' . , - :

.: . ~ . :: . . . - :, .: - . :
: . - ~
: , : : - .
, . . . . .

W091/1~7~X PCT/AU91/00141 2 ~7~3~ 30 Procedure - ~-lactoglobulin PVDF disc prepared as in (A) ls placed in the microcell. PITC Edman rea~ent ls injected as 5% PITC, 5% TEAFTA (triethylammonium trifluoroacetate) and O.Ol~ DTT in butyl chloride.
The sample electrode is charged negatively, with both proximal electrodes 128, 130 positive, l5 volts 0.6 ~A to achieve coupling of the PITC with the N-terminal amino acld. Charge ls maintained for 30 minutes while the temperature is kept at 50C. The sample cisc is washed with lO0 ,ul butyl chloride then removed from microcell and placed into ABI 470 Sequencer with preconditioned polybrene disc.
Program started at 03CPT~ step 15 (no coupling in first cycle, only washes and cleavage).

(C) ~amDle of Electrolvt~c COUD1inq and Clea~aqe Summary - ~-lactoclobulin PVDF disc placed in microcel', PITC introduced and electrolytically coupled as (B) above then polarity reversal to produce conditions for cleavage of termlnal amino acid. The sample matrix is then sequenced with ABI 470.
Procedure - ~-lactoglobulin PVDF disc prepared as in (A), placed lnto microcell, coupled with PITC as detailed for (B), washed wlth lO0 ~l butyl chlorlde then cleaved in microcell as follows: lO~ trifluoro-acetate, O.Ol~ DTT ln butyl chloride introduced to the cell. Sample electrode made positive, both proximal electrodes 128, 130 negative. Temperature maintained at 50C. 15 minutes of 15 volts, 0.6 ,uA.
Sample disc washed with lO0 ,ul butyl chloride then removed from microcell and placed into A~I Sequencer .
.. . .
.
.

.
~: .

W~91/1575X PCT/AU91/00141 ~~ 31 ~79~
with a preconditioned polybrene disc (normal 03CPTH
cycles used).

( D ) Control for ( C ) Summar~
- PITC ~ntroduced, but basic and acidic condltions not electrolytically generated, sample matrix sequenced with ABI 470.
Procedure - Exactly the same as ~C) except no electrical current used in the microcell. Same coupling and cleavage reagents, time and temperature.
( E ) ~:amDle of ~lectroDhoret~c ITC Introduct~on and l~lectrolytic CouDl~nq and Cleavaqe SummarY
- ~-lactoglobulin PVDF disc placed in micrncell, rhodamine ITC electrophoretically focused onto the sample matrix. Basic conditions electrolytically generated to promote coupling then acid conditlons to achieve cleavage. Sample matrix then sequenced with ABI 470.

Procedure - ~-lactoglobulin PVDF disc prepared as in (A) placed into microcell. Coupling achieved by ln~ecting lO mg rhodamine ITC, 5% TEAFTA, 0.01% DTT
in 5 ml butyl chloride. Sample electrode held negative 15 volts, 0.7 ,uA for 30 minutes with both proximal electrodes posltive. Temperature malntained at 50-C. Cell washed with lO0 ~l butyl chloride then cleaved as described for (C). Cell washed with lO0 ~l butyl chloride. Disc removed from microcell and placed into ABI 470 Sequencer :.: . : - : - , W091/1~75~ PCT/AII91/00141 '~7~B~
with preconditioned polybrene disc (normal 03CPTH
cycles).

The data from each of experiments A to E a~ove are depicted on Fig. 6, expressed as plcomoles, PTH-amlno acids released per cycle (l letter amino acid codes).
Three cycles of the ABI 470 were undertaken (i.e. the three N-terminal am1no acids of the ~-lactoglobulin remaining on the sample matrix were identified).

Referring to Fig. 6, reading control (A) downwards through the three cycles, the N-terminal sequence is clearly Leu-Ile-Val.
In experlment (B), the presence of Leu at cycle l indicates coupling (but not cleavage) of PITC to the ~-lactoglobulin. At cycle 3 however it is apparent that all three amino acids survived the initial electrolytic coupling reaction of PITC to L.

In experiment (C), (electrolytic coupling and cleavage), the thing to note ls the presence of a relatively large Ile figure in the first cycle. (The Leu reading merely indicates non optimal cleavage of this experimental system.) The presence of an Ile reading ln the first cycle of the remaininq amino acids on the sample matrix clearly shows that cleavage of the end terminal L has been effective. Similarly the Val readins in the second cycle, shows that Ile was the terminal amino acid.

Experiment (D), the control for experiment (C), indicates that without energizing of the electrode, truncation of the terminal amino acids does not take place.

, .
.
: ', , ' ~ -.. . ................ .
.

W~91/l~?i~ PCT/A~91/00141 3 3 2 ~
In experiment (E), as with (C) above, the presence of Ile in the first cycle and Val in the second cycle indicates that the electrophore~ic/electrolytic coupllng/cleavage successfully removed the terminal amino acid.

Although the invention has been illustrated by reference to the Edm~n degradation sequencing of a protein, the application of electrophoretic transfers of a chemical reactant to or from an i~mobilized first reactant and/or electrolytic activation of reactants may be applicable to other sequencing and/or sequential chemical reactions, such as the construction of macromolecules. Furthermore, apparatus in accordance with the invention may have a plurality of spaced storage loci by providing a plurality of electrodes spaced from the reaction locus and under the control of appropriate electric voltages.

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.

Claims (66)

CLAIMS:-

1. A method for conducting a chemical reaction, the method comprising; immobilizing a first reactant at an electrode defining a reaction locus; introducing an additional reactant to the reaction locus, electrolytically activating a reaction between the first and additional reactants; and removing remaining additional reactant and/or a reaction by-product from the reaction locus.

2. A method according to claim 1 wherein the additional reactant has an electric charge and wherein the electrode is energized to electrophoretically introduce the additional reactant to the reaction locus and to cause the electrolytic activation.

3. A method according to claim 1 wherein the first reactant is a macromolecule, wherein the reaction is a cleavage reaction to form the reaction by-product, and wherein the reaction by-product and/or the remaining portion of the first reactant is removed for analysis to determine the structure of at least a portion of the macromolecule.

4. A method according to claim 3 wherein the analysis comprises capillary electrophoresis or mass spectrometry of the reaction by-product.

5. A method according to claim 3 wherein the reaction is a sequence determination of the sub-units of the macromolecule.

6. A method according to claim 5 wherein the reaction forms a cycle within a sequential terminal degradation of the macromolecule.

7. A method according to claim 5 wherein the macromolecule is a nucleotide, polysaccharide or protein.

8. A method according to claim 3 wherein the cleavage reaction comprises two stages, a first coupling stage of the additional reactant to a predetermined portion of the first reactant and a second cleavage stage to produce the reaction by-product, at least one of the stages being electrolytically activated.

9. A method according to claim 8 wherein both the coupling and cleavage stages are, in sequence, electrolytically activated by a first polarity of energization of the electrode followed by a second polarity of energization.

10. A method according to claim 9 wherein the first reactant is a protein, the chemical reaction is a cycle in a sequential terminal degradation of the protein and wherein the additional reactant is an Edman or C-terminal thiocyanate reagent.

11. A method according to claim 1 wherein the electrolytic activation of the reaction is achieved by negative energization of the electrode inducing in situ base generation by electrolysis of a solvent or further reagent adjacent the reaction locus.

12. A method according to claim 1 wherein the electrolytic activation of the reaction is achieved by positive energization of the electrode inducing in situ acid generation by electrolysis of a solvent or further reagent adjacent the reaction locus.

13. A method according to claim 1 wherein the first reactant is immobilized in a permeable sample matrix defining the reaction locus and having a volume of 1 x 106 nl or less.

14. A method according to claim 13 wherein the volume of the sample matrix defining the reaction locus is 1 x 103 nl or less.

15. A method according to claim 14 wherein the volume of the sample matrix defining the reaction locus is of the order of 10 nl to 100 nl.

16. A method according to claim 1 further comprising the colorimetric, amperometric or spectrophotometric monitoring of the reaction locus to determine the extent of the reaction between first and additional reactants.

17. A method for conducting a chemical reaction, the method comprising; immobilizing a first reactant at a reaction locus; introducing an additional reactant to the reaction locus; allowing a reaction between the first and second reactants to produce a reaction by-product; if the by-product is not charged, imparting an electric charge to the reaction by-product: and electrophoretically removing the reaction by-product from the reaction locus.

18. A method according to claim 17 wherein the electrophoretic removal of the reaction product injects the by-product into the sample port of an analysis instrument.

19. A method according to claim 18 wherein the sample port is separated from the reaction locus by a by-product permeable membrane.

20. A method according to claim 19 wherein the membrane is an ion exchange membrane.

21. A method according to claim 19 wherein the reaction locus comprises a (1) polar, or (2) non polar medium wherein the sample port has a corresponding (1) non polar, or (2) polar medium and wherein the separation of the by-product is achieved through the hydrophobicity or hydrophilicity of the membrane.

22. A method according to claim 17 wherein the by-product immediately after the reaction is not charged and wherein a charged surfactant is introduced to the reaction locus to impart a charge to the by-product.

23. A method according to claim 17 wherein the first reactant is a macromolecule, wherein the reaction is a cleavage reaction to form the by-product, and wherein the reaction by-product and/or the remaining portion of the first reactant is removed for analysis to determine the structure of at least a portion of the macromolecule.

24. A method according to claim 23 wherein the analysis comprises capillary electrophoresis or mass spectrometry.

25. A method according to claim 23 wherein the reaction is a sequence determination of the sub-units of a macromolecule.

26. A method according to claim 25 wherein the reaction forms a cycle within a sequential terminal degradation.

27. A method according to claim 26 wherein the macromolecule is a nucleotide, polysaccharide or protein.

28. A method according to claim 27 wherein the first, reactant is a protein, the chemical reaction is a cycle in a sequential terminal degradation of the protein and wherein the second reactant is an Edman or C-terminal thiocyanate reagent.

29. A method according to claim 17 further comprising an electrolytic activation step as defined in claim 1.

30. A method according to claim 17 wherein the first reactant is immobilized in a permeable sample matrix defining the reaction locus and having a volume of 1 x 106 nl or less.

31. A method according to claim 30 wherein the volume of the sample matrix defining the reaction locus is 1 x 103 nl or less.

32. A method according to claim 31 wherein the volume of the sample matrix defining the reaction locus is of the order of 10 nl to 100 nl.

33. A method of conducting a chemical reaction, the method comprising; immobilizing a first reactant at a reaction locus; electrophoretically introducing to the reaction locus at least one charged, additional reactant;
and after reaction between the first and additional reactants removing the remaining additional reactant and/or a reaction by-product of the reaction from the reaction locus.

34. A method according to claim 33 wherein additional reactant remaining from the reaction is electrophoretically removed from the reaction locus by energizing an electrode remote from the reaction locus with an electric charge of greater magnitude than that of the reaction locus.

35. A method according to claim 34 wherein the polarity of the electrode adjacent the reaction locus is reversed to enable the remote electrode to bear the greater magnitude of electric charge.

36. A method according to claim 34 wherein the remote electrode defines a storage locus for additional reactant and wherein additional reactant can be electrophoretically reintroduced to the reaction locus.

37. A method according to claim 33 further comprising the electrophoretic removal of reaction by-products as defined in claim 17.

38. A method according to claim 33 wherein the first reactant is a macromolecule, wherein the reaction is a cleavage reaction to form the by-product, and wherein the reaction by-product and/or the remaining portion of the first reactant is removed for analysis to determine the structure of at least a portion of the macromolecule.

39. A method according to claim 38 wherein the analysis comprises capillary electrophoresis or mass spectrometry.

40. A method according to claim 39 wherein the reaction is a sequence determination of the sub-units of a macromolecule.

41. A method according to claim 40 wherein the reaction forms a cycle within a sequential terminal degradation.

42. A method according to claim 41 wherein the macromolecule is a nucleotide, polysaccharide or protein.

43. A method according to claim 42 wherein the first reactant is a protein, the chemical reaction is a cycle in a sequential terminal degradation of the protein and wherein the second reactant is an Edman or C-terminal thiocyanate reagent.

44. A method according to claim 43 wherein the first reactant is immobilized in a permeable sample matrix defining the reaction locus and having a volume of 1 x 106 nl or less.

45. A method according to claim 44 wherein the volume of the sample matrix defining the reaction locus is 1 x 103 nl or less.

46. A method according to claim 45 wherein the volume of the sample matrix defining the reaction locus is of the order of 10 nl to 100 nl.

47. A method according to claim 33 further comprising the colorimetric, amperometric or spectrophotometric monitoring of the reaction locus to determine the extent of the reaction between the first and additional reactants.

48. Apparatus for conducting a chemical reaction, the apparatus comprising an electrode disposed at a reaction locus; means to immobilize a first reactant at the reaction locus; means to introduce an additional reactant to the reaction locus; means to energize the electrode to thereby electrolytically activate a reaction between the first and additional reactants and/or to electrophoretically attract the additional reactant to the reaction locus; and means to remove from the reaction locus, remaining additional reactant and/or a reaction by-product of the reaction.

49. Apparatus according to claim 48 wherein the means to remove the reaction by-product from the reaction locus is electrophoretic.

50. Apparatus according to claim 48 wherein the immobilizing means comprises a porous matrix.

51. Apparatus according to claim 50 wherein the first reactant adsorbs to the porous matrix.

52. Apparatus according to claim 50 wherein the electrode is disposed within a reaction chamber and wherein the porous matrix substantially fills the chamber.

53. Apparatus according to claim 48 wherein the means to introduce the additional reactant include a capillary conduit.

54. Apparatus according to claim 50 wherein the sample matrix has a volume less than 1 x 106 nl.

55. Apparatus according to claim 54 wherein the sample matrix has a volume less than 1 x 103 nl.

56. Apparatus according to claim 55 wherein the sample matrix has a volume of the order of 10 nl to 100 nl.

57. Apparatus according to claim 48 further comprising (a) amperometric means to monitor current at the electrode during energization, or (b) spectrophoto-metric/colorimetric means observing the reaction locus thereby to determine the extent of the reaction between first and additional reagents.

58. Apparatus according to claim 48 further comprising an analysis instrument interface defined by a membrane which is permeable to the by-product separating the reaction locus from an analysis instrument.

59. Apparatus according to claim 48 wherein the means to remove additional reactant from the reaction locus comprises an electrode remote from the reaction locus and energizable to electrophoretically attract the remaining additional reactant.

60. Apparatus according to claim 59 wherein the remote electrode defines a reservoir for additional reactant and wherein relative voltage between the remote electrode and the electrode at the reaction locus can be adjusted to reintroduce additional reactant to the reaction locus.

61. Apparatus for conducting a chemical reaction, the apparatus comprising a reaction locus; immobilizing means for securing a first reactant at the reaction locus, means to introduce an additional reactant to the reaction locus, optionally means to impart a charge to a reaction by-product of a reaction between the first and additional reactants: and means to electrophoretically remove the by-product from the reaction locus for collection or analysis.

62. Apparatus according to claim 61 further comprising an analysis instrument interface defined by a membrane which is permeable to the by-product separating the reaction locus from an analysis instrument.

63. Apparatus according to claim 62 wherein an electrode energizable to induce the electrophoresis of the by-product is disposed on the side of the membrane remote from the reaction locus.

64. Apparatus according to claim 62 wherein the membrane is an ion exchange membrane.

65. Apparatus according to claim 62 wherein reaction medium at the reaction locus is (1) polar, or (2) non polar, where the analysis instrument has a medium which is respectively (1) non polar, or (2) polar and wherein the membrane separates the by-product through its hydrophobicity or hydrophilicity.

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66. Apparatus according to claim 61 wherein the analysis instrument is a mass spectrometer of capillary electrophoresis device.