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Prototype Transgenic Biosensor Based on Genetically Modified Plant Tissue Ae-June Wang and Garry A. Rechnitz* Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822
Genetically modified plant tissue materials offer possible advantages as molecular recognition elements in biosensor design. A prototype transgenic biosensor, using potato tissue transformed with a gene conferring &glucuronidase (GUS) biocatalytic activity coupled with fluorescence detection, is described and evaluated. Under optimal operating conditions, the transgenic sensor system gives good response to glucuronide substrate in the micromolar range and has an operating lifetime of at least 2 months at room temperature. Parallel experiments with nontransformed potato plant tissue show no glucuronidase activity. The prototypesystem described here illustrates that desired pathways can be created in plant tissue through genetic manipulation of higher plants normally lacking such pathways.
INTRODUCTION The use of plant tissue materials as molecular recognition elements is gaining increasing acceptance in biosensor design.la Principal advantages include long lifetimes, low cost, high biocatalytic activity, simplicity of construction, and morphological features of specialized plant structures.5 However, plant tissue based biosensors can be limited by interference problems due to endogenousreactions and, more importantly, the fact that higher plants often lack the rich diversity of biochemical or metabolic pathways found in microorganisms.6 Recent advances in genetic manipulation have allowed the development of transgenic plants which maintain their native properties but contain added pathways from bacterial genes.7-10 Such transgenic plant tissues may be useful candidates for biosensor design. In this paper, we describe a prototype biosensor which uses transgenic potato tissue having a substrate selectivityabsent in nontransformed plant tissues. The use of transgenic tissue materials does not, of course, necessarily overcome general problems of fragility and variability often associated with tissue-based biosensors. Figure 1illustrates the general principle of our approach. A foreign gene, in this case, @glucuronidasegene (GUS) from (1)Arnold, M. A.; Rechnitz, G. A. In Biosensors: Fundamentals and Applications; Turner, A. P. F., Karube, I., Wilson, G. S., Eds.; Oxford University Press: New York, 1987; pp 30-59. (2) Navaratne, A.; Rechnitz, G. A. Anal. Chim. Acta 1992,257,59-66. (3) Wang, J.; Naser, N. Anal. Chem. 1992,64, 2469-2471. (4) Sidwell, J. S.; Rechnitz, G. A. Biotechnol. Lett. 1985, 7,419-422. (5) Sidwell, J. S.; Rechnitz, G. A. Biosensors 1986, 2, 221-228. (6) Rechnitz, G. A. Science 1981,214, 287-291. (7) Jefferson, R. A.; Burgess, S. M.; Hirsh, D. h o c . Natl. Acad. Sci. U.S.A. 1986,83, 8447-8451. (8) Jefferson, R. A.; Kavanagh, T. A.; Bevan, M. W. EMBO J. 1987,
6,3901-3907. (9) Quandt, H.; Broer, I.; Puhler, A. Plant Sci. 1992,82, 59-70. (10) Vardi, A.; Bleichman, S.; Aviv, D. Plant Sci. 1990, 69, 199-206. 0003-2700/93/0365-3067$04.00/0
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Flgure 1. Preparation of transgenic plant tissue materials for use as molecular recognition elements in biosensor design.
Escherichia coli, is sequenced and cloned into Agrobacterium tumefuciens with DNA manipulation technology and transformed to the potato plant to express GUS activity as a reporter gene for gene fusion analysis. GUS in transgenic potato plants is primarily concentrated in the vascular ring and involves the hydrolytic cleavage of a variety of p-glucuronides to generate an alcohol and the glu~uronate.~ The analysis of glucuronides is of some significance in clinical chemistry as glucuronide is believed to be capable of detoxifying poisonous hydroxyl-containing substances in urine.11 Thus, such transgenic plant tissue may be suitable as molecular recognition elements for biosensor design. While electrochemical detection of reaction products in transgenic plant tissues may be possible, our effort has focused on the development of a transgenic fiber-optic biosensor. In this study, transgenic potato plant tissue with GUS activity has been immobilized in a reactor, with the generated fluorescence product being detected in a flow injection arrangement. The substrate 4-methylumbelliferyl p-D-glucuronide (MUG) was used for preliminary study, with the generated product 4-methylumbelliferone (MU) being measured fluorometrically at 450 nm upon excitation at 360 nm. The construction of tissue reactors, response properties, and the characteristics of the prototype transgenic biosensor will (11)The Merck Index, 11th ed.; Merck Rahway, NJ, 1989; p 701.
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be detailed below. It will be seen that the results are sufficiently promising to indicate that the development of a discrete sensor may be possible with further development. EXPERIMENTAL SECTION Apparatus and Reagents. The tissue reactor was placed between the pump and the fluorescence detector with carrier buffer being pumped by a HPLC pump (Shimadzu,LC-SA,Kyoto, Japan) through a Lo-Pulse dampener (Scientific Systems, Inc., Model LP-21 to reduce pressure fluctuation. The analyte was injected using a Rheodyne injectorwith a 20-pL sample loop and carried by the carrier buffer to the tissue reactor. A water jacket surrounded the tissue reactor, and a water bath (Fisher, Model 900) was used to control the temperature of the reaction. The substrate was converted to the fluorescent product by the GUS in the reaction chamber. A fluorescence detector (Jasco,Model 821-EP, Tokyo, Japan) was located downstream of the reactor for the detection of products. A chromatointegrator (Hitachi, Model D-2500) was used to record and analyze the fluorescence signals from the detector. 4-Methylumbelliferone (MU), 4-methylumbelliferyl B-D-glucuronide (MUG),5-bromo-4-chloro-3-indolyl-~-~-glucuronide (Xgluc), and glass beads (150-212 pm) were purchased from Sigma Chemical Co., St. Louis, MO. Transgenic potato tubers were gifts from William R. Belknap, United States Department of Agriculture, Albany, CA. The regular Russet potato used for blank experimentswas purchased locally. The glass column (OM6310) with a water jacket (OM-6330) used for the tissue reactor was purchased from Chromatographic Techniques Inc., Apple Valley, MN. Procedures. Genetic Transformation of Potato Plant. Genetic transformation of potato plant was accomplished using Agrobacterium tumefaciens, which carries a disarmed Ti plasmid pAL4404 and the binary vector PBI121. Two genes, GUS driven by the cauliflower mosaic virus 35s promoter and neomycin phosphotransferase driven by nopaline synthase promoters in the plant cell, were carried by this vector PBI121.7.8 Agrobacterium was grown in agar and selective medium and used for the following plant inoculation. A thin slice of potato microtuber was placed in a petri plate with a medium, and a small amount of Agrobacterium was transferred to the cut surface of the tuber surface for the transformation of the GUS gene. The tuber was grown in a tissue culture with selective medium. The above operational procedures and experimental conditions have been detailed in the paper by Belknap et al.I2 Histochemical Assay. The transgenic potato tuber was planted in regular potting soil and watered every other day. The tuber started to sprout after 1 month, and the young potato plant grew to a height of 10 cm within 2 weeks after sprouting. The GUS activity in the plant was visualized by staining the plant tissue with a histochemical substrate 5-bromo-4-chloro3-indolyl-~-~-glucuronide (X-gluc). The buffer used in the staining procedure was "GUS buffer", which consisted of 5.34 mg of monobasic sodium phosphate, 8.70 g of dibasic sodium phosphate, 0.22 g of potassium ferrocyanide, 0.16 g of potassium ferricyanide, 0.37 g of ethylenediaminetetraacetic acid (EDTA), and 1mL of Triton X-100in 1L of deionized water. The staining solution was composed of 1 mg of X-gluc and 20 pL of dimethylformamide dissolved in 1 mL of GUS buffer. A thin sliceof the tissue from differentsections of transgenic plant (stem, petiole, leaf, root, etc.) was placed in a microcentrifugetube with 50 pL of the staining solution. The tissue was incubated in the stainingsolution overnight.13 The stained tissue was then rinsed with ethanol for 10 min. Tissue Reactor Construction. The transgenic plant tissue for the construction of the tissue reactor was obtained from a young potato plant. A small portion of stem and petiole was removed from the plant and chopped into small pieces with a razor blade. The chopped tissue was then transferred to a mortar with a small portion of 0.1 M phosphate buffer (pH 7) and ground (12) Ishida,B. K.; Snyder,G.W.; Belknap, W. R.Plant CeZZRep. 1989, 8,325-32a. (13) Navaratne, A. N. Ph.D. Dissertation, University of Hawaii at Manoa, HI, 1992.
with a pestle. Glass beads were then mixed with the ground tissue in the mortar in the ratio of approximately 1 to 3. The mixed tissue and glass beads were transferred to the glass column and packed tightly. A small amount of glass wool was used to block both ends of the column. Phosphate buffer (0.1 M, pH 7.5) was passed through the tissue reactor for 30 min with 2 mL/min flow rate. The nontransgenic tissue reactor was fabricated in the same manner. The stem and petiole from the regular potato plant were used instead of the transgenic plant tissue in the tissue reactor. Sensor Characterization and Construction of Calibration Curve. Flow injection analysis (FIA) was operated using 0.1 M phosphate buffer flowing at 2 mL/min. Unless stated otherwise, the tissue reactor was maintained at 25 "C. The MUG standards were prepared by diluting 1mM of MUG stock solution with pH 7.5 phosphate buffer. The fluorescence signal peak resulting from the MU produced was integrated and the peak area calculated as the sensor response. The calibration curve was constructed by plotting the sensor response versus the corresponding MUG concentration. The analysis time was defied as the period of time taken from the initialincrease in fluorescence signal to the return to baseline.
RESULTS AND DISCUSSION GUS activity in plants can be visualizedusing histochemical methods. Various sections of young and mature transformed plants were stained with X-gluc, and the resulting deep blue color indicated the distribution of GUS activity in the tissue. Enzyme loading cannot be accurately determined from this staining experiment because the color intensity in the tissue section also reflects cell numbers per unit area.8 Nevertheless, it is clear that the GUS gene is expressed throughout the plant including stem, petiole, leaves, root, callus, and buds from either young or mature plants with highest concentration in the vascular bundle of the young plant. The stem and petiole were used directly for the construction of the tissue column without further dissection of the vascular ring. Staining of the nontransformed potato plant showed no blue color even after 48-h exposure, confirming the lack of endogenous GUS activity in the higher plant. Several arrangements for the tissue reactor were investigated in order to achieve the highest enzyme-catalyzed conversion in the reaction chamber. All of the experiments performed for evaluating the packing methods were operated at 25 "C with pH 7.5 phosphate buffer. The injection of 50 pM MUG was used to compare the intensity of the signals generated from the fluorescent product. Optimal results were obtained with an arrangement where plant tissue segments were first chopped and ground, and then mixed with glass beads (3:l ratio), before packing in the reaction column. Such an arrangement provides sufficient contact between substrate and tissue without excessive pressure buildup in the reactor. Before optimization and characterization of the transgenic biosensor, a blank experiment using MUG was performed with a nontransformed tissue reactor. No signal was detected with this nontransformed system. Therefore, we can conclude that the generated fluorescence signal is from the GUS enzymatic reaction rather than from endogenous reactions in the potato plant. Sensor response is affected by several parameters such as the flow rate, the pH of the carrier buffer, and the temperature of the reaction chamber. Figure 2 shows the response to injections of 50 pM MUG at various flow rates ranging from 0.5 to 3 mL/min. This flow rate study was operated in pH 7.5 phosphate buffer at 25 "C and demonstrates that the slower carrier flow produces greater fluorescence response from the generated product. As the flow rate increases, the residence time of the substrate in the reactor is shorter and
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the enzymaticconversionefficiencydecreases with a decrease in generated fluorescence products. Although slower flow gives higher responses, sample analysis is also longer. The analysis times for increasing flow rates from 0.5 to 3 mL/min are 30,20, 16,12,9, and 8 min. Analysis time in this transgenic tissue column is longer than in the common immobilized enzyme reactors,14 perhaps because of longer path lengths or because of substrate and/or product diffusion across the wall of the plant cells employed. Therefore, a compromise is necessary between the sensitivity of the system and the analysis time. A flow rate of 2 mL/min was chosen as the optimal flow rate for subsequent experiments. Temperature is a key factor for most enzymatic reactions. In most cases, the activity of the enzyme increases with increasingtemperature. A similar tendency was observed in this transgenic plant sensor with the results demonstrated in Figure 3. The examined temperature ranges were from 20 to 45 "C.Results indicate that sensor response is higher at higher temperatures within the tested temperature range. The response decreases at temperatures above 45 "C,probably due to damage to the structure and metabolism of the plant as well as denaturation of the enzyme in the cell. The baseline of the recording also fluctuated at higher temperature. Although the sensor response at 37 "C is 150% that at 25 "C, (14) Bowers,L.D.; Johnson, P. R. Biochim. Biophys. Acto 1981,661, 100-105.
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response at 25 "C is adequate for the measurements in this study and also allows ease of experimental protocol. Therefore, 25 "C was chosen as the operational temperature for all subsequent experiments. Figure 4 represents the effect of pH between 6 and 9 on the response of the system. There are two factors which determine the overall sensor response in this system, i.e., the influence of pH on the enzyme activity and the effect of pH on the generated fluorescence product. The optimal pH for the glucuronidaseenzyme in free form is appro~imately6,8.~~J~ However,the fluorescenceintensity of MU is stronger in basic solution. The decrease in sensor response at the more basic pH is thus thought to be the result of decreasing enzyme activity. Therefore, pH 7.5 gives the highest response as a compromisebetween these opposing factors. The system was operated at pH 7.5 throughout this study. A calibration curve obtained under these conditions is shown in Figure 5. The intergrated fluorescence intensity generated is directly proportional to the concentration of the anal* up to a concentration of 5 mM. After that, the signal deviates from linearity,possibly due to fluorescenceinhibition (15) Barman, T. E. E n z y m e Handbook; Springer-Verlag Berlin-Heidelberg: New York, 1985; Vol. 11, pp 590-591. (16) Mum,B. U.;Doe,R. P.;Seal, U.S. J. Biol. Chem. 19611, 240, 2811-2816.
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of MU at higher concentrations.17 The regression coefficient of the linear plot for the concentration range from 1to 100 pM is 0.9996, and the detection limit for the transgenic tissue sensor is, thus, below 1 pM. It can be seen (Figure 5) that the nontransgenic tissue gives no response over the range tested under the same experimental conditions. The time stability of the system was evaluated by making triplicate measurements each day with 50 pM MUG under optimized conditions. The tissue reactor was stored in pH 7.5 phosphate buffer at room temperature when not in use. The sensor retained 98 % of the original response after a period of 2 months. We believe the lifetime of the sensor could be extended even further if the tissue reactor is stored at 4 "C. The relative standard deviation for the measurements (n = 3) is approximately 2 % The reproducibility of different tissue reactors was also examined and the relative standard deviation is about 5% (n = 3). The relatively slow response time (12 min at 25 "C) is the major drawback of this system. We believe this problem can be overcome by immobilizing the transgenic plant tissue at the tip of a fiber-optic probe. At the present time the cost of transgenic plant materials represents a practical limitation of the proposed concept, but
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such coats are expected to decline drastically in the future as transgenic strains become more widely available.
CONCLUSION The prototype system described here is intended to demonstrate the concept of using transgenic materials as molecular recognition elements in biosensor design. In the case of plant tissues, this approach will permit a much richer range of possible pathways through the insertion of genes not naturally found in plants while the morphological advantages of higher plant structures areretained. While it will be obvious that extraordinary levels of selectivity could be achieved using this technology, we also envision a useful technique for the investigation of the transgenic materials themselves.
ACKNOWLEDGMENT We thank William Belknap for providing the transgenic potato tubers. Financial support by National Science Foundation Grant CHE-9216304 is greatly appreciated.
RECEIVEDfor review April 19, 1993. Accepted July 29, 1993.'
(17) Skoog,D.A.AinciplesofInstrumentalAnulysis,3rded.; Saunders College Publishing: New York, 1985; Chapter 8.
Abstract published in Advance ACS Abstracts, September 15,1993.
