498
Anal. Chem. 1993, 65, 498-499
Detection of Electrophore-Labeled DNA and Albumin by Gas Chromatography: Labile Amide Electrophoric Release Tags Samy Abdel-Baky; Kariman Allam, and Roger W. Giese' Department of Pharmaceutical Sciences in the Bouve College of Pharmacy and Health Professions and the Barnett Institute, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115 Molecular labels such as radioisotopes and fluorescent dyes are widely used for detection purposes in analyticalchemistry. For example, a DNA probe or antibody may be labeled with one of these compounds. We are interested in using electrophores as molecular labels. An electrophore is a compound that combines with a low-energy electron in the gas phase. Thus electrophores are commonly detected by gas chromatography with electron capture detection (GC-ECD), and by GC with detection by electron capture negative ion mass spectrometry (GC-ECNI-MS). Subattomole amounts of strongelectrophores have been detected by these techniques. Polyhalogenated molecules like carbon tetrachloride and lindane are typical electrophores. While the high sensitivity of electrophores is important, our basic motivation for using electrophores as molecular labels is their ease of providing high multiplicity. Both GCECD and GC-ECNI-MS can measure many electrophores with each injection. Thus many different DNA probes, for example, eachlabeled separatelywith a different electrophore, might then be combined for use as a single reagent that could provide multiple hybridization assays simultaneously. Or a mixture of electrophore-labeled antibodies might be used similarly to detect many antigens. The specificity in detection would derive from the biorecognition event (ag. of an electrophore-labeled antibody recognizing an antigen), and each such event in a given, multievent assay would be monitored by a different electrophore. Here we demonstrate the detection of electrophore-labeled DNA and albumin, as model compounds, in a combined sample.
EXPERIMENTAL SECTION Equipment. A Varian 3700 gas chromatograph fitted with an electron capture detector (maintained at 310 "C) was used (Varian, Sugar Land, TX). For the injections, a rotary valve (which avoided the problem of septum bleeding at the injector temperature of 300 "C) from a Varian 1097 on-column capillary GC injector was connected to a Varian flash vaporization injector body. The latter contained a glass insert (13 cm X 6 mm, length X 0.d.). A hole (4-mm i.d.) at the inlet tapered to a 2-mm i.d. after 3 cm. The bottom of the upper, larger bore was packed with 32 mg of acid-cleaned glass wool onto which 1-pLinjections were made through a 11.5-cm stainless steel needle connected to a syringe. The chromatographic column was a 0.32-mm-i.d. X 7-m Quadrex 007 column (5% phenylmethylsilicone; Quadrex, New Haven, CT), 5-pm film thickness. After sample injection, the column was held at ita initial temperature of 50 "C for 3 min, then programmed at 50 "C/min to 150 "C, and held at this temperature for 5 min. Nitrogen flow through the 50 "C column was 3 mL/min. Synthesis. N-(Trichloroacety1)-p-aminobenzoicAcid Nhydroxysuccinimide Ester (1). p-Aminobenzoic acid (1g, 7.3 mmol) and 7 mL (32.8 mmol) of trichloroacetic anhydride were refluxedfor 0.5 h. More anhydride (3mL) was added, and heating was continued for 16 h. Water (15 mL) and ethyl acetate (30 mL) were added, and after shaking, the separated organic layer + Present address: BASF Corp., Research Triangle Park, NC 277093528. To whom correspondence should be addressed. (1) Corkill,J. A.; Joppich, M.; Kuttab, S. H.; Giese, R. W. Anal. Chem. 1982,54, 481-485. (2) Abdel-Baky, S.; Giese, R. W. Anal. Chem. 1991, 63, 2986-2989.
0003-2700/93/0385-0498$04.00/0
was dried (NazSOd) and rotary evaporated to give N-(trichloroacety1)-p-aminobenzoicacid as yellowish-white crystals (1.95 g, 95%), which was a single spot by silica TLC (ethyl acetate/ hexane, 2/3). This intermediate (290 mg, 1.03 mmol) was dissolved in 5 mL of dimethylformamide (DMF), and the temperature was raisedto 70 "C. N,N-carbonyldiimidazole(144 mg, 0.90 mmol) was added, and 70 "C was continued until COz evolution ceased (30 rnin). N-Hydroxysuccinimide (NHS, 102 mg, 0.09 mmol) was added, heating was discontinued, and the reaction mixture was stirred for 17.5h. The solvent was removed under high vacuum, and the addition of 15 mL of 2-propanol gave the product as a white precipitate (304 mg, 81%) which was a single spot by silica TLC (ethyl acetate/hexane, 2/3), mp 258 "C. IR (KBr): 1790(CChCO)and 1705(N-CO) cm-l. 'H NMR (acetone-de): 6 2.96 (s,2CHz), 7.91 (d, 2 H), and 8.1 (d, 2 H). MS (EI): mlz 264,230,200,and 146. MS (NCI): m/z 378 (M-1. Anal. Calcd for C13H&hNz06: C, 41.13; H, 2.39; C1, 28.02; N, 7.38. Found: C, 40.98; H, 2.41; C1, 27.85; N, 7.61. N'- (Trichloroacryloy1)-p-aminobenzoicAcid N-hydroxysuccinimide Ester (2).Trichloroacrylic acid (500 mg, 2.85 mmol; Alfa, Danvers, MA) was added to 7 mL of SOC12, and the mixture was refluxed for 6 h. After cooling, 5 mL of benzene was added and volume was reduced by two-thirds on a rotary evaporator. This step was repeated four times, until most of SOClz was evaporated. Acetonitrile (3 mL) was added followed by a suspension of 383 mg (2.80 mmol) of p-aminobenzoic acid in 3 mL of acetonitrile. After 30 rnin of stirring, TLC showed the disappearance of most of the starting material and the presence of a new compound with a higher Rfvalue, which was purified by preparative silicaTLC (ethyl acetate/hexane/aceticacid, 2/3/ 0.05) yielding (trichloroacryloy1)-p-aminobenzoicacid as a white powder (729 mg, 89% yield). MS (EI): m/z 293 (M+). Dicyclohexylcarbodiimide(10 mg, 0.05 mmol) was added as a solid to a stirred suspension of this intermediate (14 mg, 0.047 mmol) and NHS (5.98 mg, 0.05 mmol) in 3 mL of dry CHzClz at 0 "C. The reaction mixture was stirred under Nz for 3 h and allowed to come to room temperature. The insoluble dicyclohexylurea was filtered, and rotary evaporation of the filtrate yielded a white solid that was purified by preparative TLC (ethylacetate/hexane/ acetic acid, 2/3/0.05), giving the product as a white powder (12 mg, 65%). lH NMR (acetone-de): 6 3.00 (s, 2 CHz), 7.96 (d, 2 H), and 8.81 (d, 2 HI. MS (EI): m/z 390 (M+) and 276 (base peak). Anal. Calcdfor CI&I&~NZO~: c , 42.94; H, 2.32; C1,27.16; N, 7.16. Found C, 43.06; H, 2.52; C1, 27.45; N, 6.94. 1-NH-DNA was prepared by slowly adding 15mg (0.038 mmol) of N-(trichloroacety1)-p-aminobenzoicacid NHS ester in 700 pL of formamide/DMSO, 4:3, v/v, to a solution of 2 mg of 1,8diaminooctyl-substituted DNA (DAO-DNA prepared as described in ref 3) in 800 pL of 0.1 M potassium phosphate, pH 8 (buffer A). The resultant cloudy solution (precipitation of the DAO-DNA by the organic solvent) was stirred at room temperature for 18 h. The reaction mixture was centrifuged, and the clear supernatant was passed through two PDlO columns (Pharmacia, Piscataway, NJ) using 0.01 M buffer A, followed by lyophilization. 1-Albumin was prepared by reacting 2 mg (0.02pmol)of bovine serum albumin (dissolved in 1.8 mL of a 0.1 M potassium phosphate buffer, pH 8/DMSO, 1:l)with N'-(trichloroacety1)p-aminobenzoicacid NHS ester (16.6 mg, 0.04 mmol). The latter was added in solid portions over 15 min, followed by stirring for 18 h at room temperature. This solution was centrifuged, and the clear supernatant was passed through two PDlO columns using0.01M buffer A, followed by lyophilization. Protein analysis (3) Cecchmi, D. J.;Guan, K. L.; Giese, R. W. J . Chromatogr. 1988,444, 97-106.
0 1993 American Chemlcal Soclety
ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993
B
A
0
4
0
4
TIME, minutes Flgure 1. (A) Detection of 1-DNA and 2-albumin by GC-ECD. A 1-pL aliquot of water containing 331 ng of 1-DNA (gMng the peak for CCi3H), 147 ng of 2-albumin (gMng the peak for CCI,CCIH), and 334 ng of polylysine was injected over a 20-s period into the instrument. (B) Blank injection of the same quantities of DNA and albumin lacking the electrophore labels, along with polylysine.
(BCA test, Pierce Chemical Co.) and amino group analysis (trinitrobenzenesdfonate test) were used to determine the number of the primary amino groups on the albumin that were modified. 2-Albumin was prepared and characterizedsimilarly.
RESULTS AND DISCUSSION Our scheme for detecting electrophore-labeled DNA and albumin by GC-ECD is shown here. 0
E-AM+LN> +
0
Macromolecule DNA or Albumin )
,
I
1) E J CCI, 2)
(”2-
E I CClpCCl
E - ~ ~ N H + cMacromolecule L
I
E-
GC-ECD
H
The reactive, electrophore-containingreagents 1 and 2 are new types of ‘electrophoric release tags”, possessing a hydrothermally-labile amide as a novel release group. Previously glycol, olefin,and methionylamide release groups were demonstrated for eledrophoric release tags.4 Compounds 1 and 2 were prepared starting from p-aminobenzoic acid. DNA was first transaminated with diaminooctane, giving ‘“2-DNA”, which in turn was labeled with 1 in aqueous buffer yielding ‘1-NH-DNA”. Albumin was directly reacted with 1 under similar conditions, giving “1albumin”, and also “2-albumin” was prepared. The number of CCb groups on 1-albumin was 51 (correspondingtolabeling 82% of the primary amino groups), and there were 32 CHClCClz groups on 2-albumin. Injection of an aqueous solution of 1-NH-DNA and 2-albumin into a GC-ECD having a hot (300“C) injection port and a warm (50 “C) capillary column gave peaks for (4) Abdel-Baky, S.; Klempier, N.; Giese, R. W. Tetrahedron 1990,46, 5859-5880. (5) Senft, V. J. Chromatogr. 1985,337, 126130. (6) (a) Grob, K. J. Chromatogr. 1984,299, 1-11. (b) Dingyuan, H.; Jianfel, T. Anal. Chem. 1991, 63, 2078-2080. (7) Church, G. M.; Kieffer-Higgins, S. Science 1988,240,185-188. (8) Kricka, L.J., Ed.Nonisotopic DNA Probe Technique; Academic Press: San Diego, 1992. (9) Arlinghaus, H. F.;Thonnard, N.; Spaar, M. T.; Sachleben, R. A.;
Larimer, F.W.; Foote, R. S.; Woychik, R. P.; Brown, G. M.; Sloop, F.V.; Jacobson, K. B. Anal. Chem. 1991,63, 402-407.
499
CC4H and CC12CClH, respectively, as shown in Figure 1A. No peaks were observed a t these positions when a control sample of DNA and albumin was injected, as seen in Figure 1B. We prepared compounds 1 and 2 to function as electrophoric release tags stimulated by the observation, made by others: that trichloroacetic acid thermally decomposes in hot water to form chloroformand COZ. As a step toward the development of 1 and 2, we observed that their immediate synthetic precursors (the corresponding substituted aminobenzoic acids) were each stable in water but formed peaks for chloroform (93% yield) and trichloroethylene,respectively, when injected into the GC-ECD, as did injections of trichloroacetic and trichloroacrylic acids, respectively. The latter acids may be formed as intermediates when the above electrophore-tagged macromolecules are injected into the instrument. Interferences from pyrolysis of these macromolecules are minimized since the GC column is heated only slightly,and the ECD is selective for electrophores. The steam that is generated in the injector by the aqueous sample moves ahead of the CC13H and CCl2CClH in the GC column, as observed previously when CCSH in water is injected into a GC-ECD.6 We found that as little as 2 X 10-l6mol of 1-albumin could be detected (data not shown). The absolute response for duplicate injections varied I l O % . A linear response was observed up to at least 6 X mol for this conjugate (r = 0.99). Slow injections (20 8 ) were made in order to more completely expose the samples to the high temperature of the injection port: this increased the response. Initially,there was no response for 1-DNA. This was overcome by adding polylysine to the sample prior to injection. The intent was to aggregate the 1-DNA to increase its residence and thereby the release of CC13H in the injection port, but the true mechanism by which polylysineincreased the release of CChH is unknown. It should be possible to prepare many compounds like 1 and 2, each having aunique electrophoricgroup, E,tofunction similarlyin our system. One such compound could be seleded to function as an internal standard to improve the precision. The use of GC-MS instead of GC-ECD could expand the number of hydrothermal release products, E-H, that could be detected with each injection and also the speed of the determination. Such multiplicity is anticipated to increase the analyticalusefulnesa of DNA probes and related reagents. For example, it can be important to subject a given sample of DNA to multiple hybridization reactions? Since current labels for DNA probes have limited multiplicity,8 such hybridizations now must be done sequentially, which is tedious. The use of a cocktail of electrophore-labeledDNA probes could reduce the time and effort involved. However, specialized equipment will be necessary to gain these advantages. Stable isotopes are also of interest as labels for DNA probes to increase labeling multipli~ity.~ In our future work we will test this concept further by increasingthe multiplicity of the electrophoresand conducting DNA hybridization assays with electrophore-labeled DNA probes. The relatively small size and moderate polarity of the electrophore labels should help to minimize the background signal due to nonspecific binding of the probes in such an assay.
ACKNOWLEDGMENT This work was supported by DARPA Contract N0001982-K-0811 administered by the Office of Naval Research, a Science and Engineering Award from Dupont,and N M Grant HG00029. Contribution No. 556 from the Barnett Institute.
RECEIVED for review July 1992.
6, 1992. Accepted November 9,
