Anal. Chem. 1995,67,4229-4233
A Holographic Sensor for Proteases Roger B. Millington,* Andrew 0. Mayes, Jeff Blyth, and Christopher R. Lowe Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT U.K.
The use of holographic elements as biochemical sensors is suggested and exemplifiedwith a device for monitoring protease activity. A quantitative optical response of a holographic element constructed in gelatin is demonstrated for a range of trypsin concentrations down to 25 nM with a response time within 20 min. These data demonstrate the principle for a general protease sensor which has particular relevance to the measurement of trypsin activity below normal physiological duodenal levels. The holographic devices respond with a change in wavelength (color) and/or a change in brightness. The possibilityof creatinga famih,of spedic, reagentless, lowcost holographic sensors with direct visual output is outlined.
h a n n et al.' measured normal physiological levels of trypsin in duodenal fluid and in feces as an aid to diagnosis of pancreatic disease. The concentration of trypsin in duodenal fluid was found to be in the range 420-2680 pg/mL (18-115 pM), while the concentration of trypsin in feces is much lower, typically >30 pg/ g, (typically diluted to >1nM). The assay used was based on trypsin hydrolysis of p-toluenesulfonyl-L-arginine-methylester (TAME)substrate and monitored by titration against liberated H+. During similar studies, other worker^^,^ found similar levels of trypsin in feces: For example, Barbero et al.2 showed that the stool trypsin level was generally at least 1 order of magnitude lower in samples from children suffering from cystic fibrosis compared with levels from healthy children. Crossley et al.4 described a method for screening for cystic fibrosis using a chromogenic synthetic substrate, sodium benzoyl-DL-arginiiek nitroanilide (BAPNA) for trypsin in stools. A much simpler but only semiquantitative method of measuring duodenal or fecal trypsin by visual observation of the degradation of a gelatin film has been described by Demetriou et a1.5 and gives a 30 min response by incubation at 30 "C. In the holographic world, the action of trypsin on gelatin-based structures has been used to create a surface relief hologram by etching the surface of a tanned emulsion: the enzyme degrades the gelatin more completely where cross-linking has been limited. Trypsin has also been used to change the color of parts of a (1) Amman, R W.; Tagwercher,E.; Kashiwagi, H.; Rosenmund, H. Am. 1.Dig. Dk. 1968,13,123-146. (2) Barbero, R W.; Sibinga, M. S.; Marino, J. M.; Seibel, RAm. /. Dk. Child. 1966,112, 536-540. (3) Haverback, B. J.; Dyce, B. J.; Gutentag, P. J.; Montgomery, D. W. Gasfroenterologv, 1963,44, 588-597. (4) Crossley, J. R; Benyman, C. C.; Elliott, R B. Lancet 2 1977,1093-1095. (5) Demetriou, J. A; Drewes, P. A; Gin, J. G.: Enzymes In Clinical Chemistry: Pn'nciples and Techniques; Henry, R J., Cannon, D. C., Winkelman, J. W., Eds.; Harper and Row: Hagerstown, MD, 1974. 0003-2700/95/0367-4229$9.00/0 0 1995 American Chemical Society
reflection hologram by weakening the structure prior to exposure.6 Holograms or, more specifically, Bragg reflectors, have been employed as sensors for physical parameters, often pressure and temperature and commonly in a fiber optic f ~ r m a t . ~ The ~* response of gelatin Bragg holograms to humidity has also been described by other workers! Several workers have described specifc biosensors in the form of surface gratings made from antibody or antigen material,10-12 although instrumentation is required for signal analysis. This paper describes the use of a gelatin-based optical hole gram, specifically a Bragg reflector, as a quantitative sensor element for measuring protease activity, the response being a change in color (wavelength) or brightness created when trypsin cleaves at peptide bonds adjacent to the arginine and lysine residues of gelatin and causes swelling of the hologram. The concept of the technique as a low-cost, reagentless, sensor for enzymes in general is introduced. In this paper we report experiments that define calibration curves for enzyme activity together with data on reproducibility. The potential for a sensor with a direct visual response and no instrumentation is indicated. THEORY
The formation of interference fringe planes in a photosensitive material and subsequent processing to record them as a distribution of refractive index is The spectral reflected response of a simple reflection hologram to incident white light is a narrow-band peak with a peak wavelength given by the Bragg relation
where n is the average refractive index of the material, D is the separation of the fringe planes, and 0 is the angle of incidence of the illuminating light beam within the medium. It is clear from eq 1that a change, dD,in fringe plane separation gives the same fractional change, Mpk, in peak wavelength response, expressed as (6) Walker, J. L;Benton, S. A. US. Patent 4986619, January 22, 1991. (7) Kersey, A D.; Berkoff, T. A; Morey, W. W. Opt. k f f1993, . 18, 13701372. (8) Meltz, G.; Morey, W. W.; Glenn, W. H.; Farina, J. D. O M Tech. Dig. 1988, 2, 163-166. (9) Spooncer, R C.; A-Ramadhan, F. A; Jones, B. E. Inf.J Opfoelectron. 1992, 7, 449-452. (10) Glanz, J. R&D Mag. 1993,(March), 51. (11) Nicoli, D. F.; Eliigs, V. B. European Patent Application EP-A3-0167335, January 8,1986. (12) Stewart, W. J. International Patent Application WO-A1-9009576,August 23, 1990. (13) Saxby, G. Practical Holography, 2nd ed.: Prentice Hall: Englewood Cliis, NJ, 1994.
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Any physical, chemical, or biological reaction that alters the fringe separation will give a measurable wavelength change. Another response that can be monitored is the brightness (the reflectivity). The fringe planes represent the high index peaks of a sinusoidal variation in index throughout the medium and can be thought of as a series of equally spaced partial reflectors. The reflectivity of the assembly of fringes is the sum of all the partial Fresnel reflectivities. If fringes are disrupted by degrading the support medium, then each fringe will tend to scatter rather than reflect light. If the support medium is gradually removed into solution, the fringes will be lost and the net reflectivity will decrease. Furthermore, if the depth of modulation of the refractive index is altered, then partial reflectivities will change, and as fringes are removed, broadening of the peak as fringes is observed. Any (bio)chemical reaction that causes the holographic material to be removed into solution or that causes the modulation depth of refractive index to be reduced is expected to reduce reflectivity.
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Hologram Construction. Agfa 8E75HD film was incubated in a borate buffer solution 112.5 mM borax/HBr, pH 8.1, containing 0.025% (w/v) NaNd for at least 12 h in order to ensure a pHcontrolled equilibrium of the emulsion in the swollen state and mechanical stability of the emulsion during exposure such that subsequent replay under similar conditions is in an easily measurable visible part of the spectrum. A reflection phase hologram was constructed under buffer at room temperature by exposure of the preswollen film to a beam of HeNe laser light at 633 nm. The optical layout shown in Figure 1was designed to provide a "clean" near-flat intensity profile over a 40 mm x 40 mm piece of film in order that the subsequent small pieces (15 mm x 5 mm) cut from it have holograms with similar physical structure and chemical properties and, consequently, reduced sample-to-sample variation in response. Exposure was followed by development and rehalogenation bleaching using standard holographic meth~dology.'~After processing, the hologram was either stored wet in buffer until use or dried and stored in a sealed plastic bag. In this form, it could be kept for at least several months. When the dried film was rewetted, it had to be reincubated in buffer for at least 12 h before use. Experimental Method. The film was cut into 15 mm x 5 mm strips and each strip placed inside a standard 4 mL polystyrene cuvette containing 1 mL of borate buffer. The temperature was controlled at 25 f 0.5 "C over the period of each test by using a modified spectrophotometer cuvette holder with recirculated water provided by a heated/cooled bath. A miniature 4 mm long x 1 mm diameter magnetic stimng bar was placed in the cuvette to ensure that mixing was rapid and analyte presentation was similar from test to test. The optical arrangement for direct observation of the reflected spectrum of the hologram is shown in Figure 2. The hologram under test was illuminated with a converging beam of broad-band light (tungsten filament halogen), which was directed back via the beam splitter and lens to the 25 pm slit of the crossed Czemy-Tumer spectrograph (LOTORIEL MULTISPEC). The spectrum was detected and stored by an LOT ORIEL INSTASPEC IV CCD camera and software. Smoothing and peak detection software was written using the code 4230 Analytical Chemistry, Vol. 67, No. 23, December 7, 7995
HAGHETICSTIRRER
Figure 2. Reflection spectrometer for measuring the spectral response of a reflection hologram to a white light, tungsten/halogen source.
specilic to the detection system and was applied to the stored spectra in order to determine the wavelength and reflectivity at peak reflected intensity. A background spectrum was recorded from a piece of film which had been subjected to exposure without fringes and processed. This was stored and subtracted from subsequent test spectra using the built-in software. A reference spectrum was provided by using an aluminized mirror in place of a test hologram in the cuvette: the spectrum was stored and used to correct for illuminant spectrum and to calculate reflectivity. A series of trypsin (Type I bovine pancreas Sigma T-8003) solutions were prepared by dissolving the solid in 1 m M HCl. For each test, a new hologram was used and the stability of the spectrum monitored for temperature and buffer equilibration over at least 15 min prior to addition of 10 pL of a known concentration of trypsin to the 1mL in the cuvette. Spectra were recorded at 1 min intervals over 30 min and were stored prior to analysis. Figure 3 shows a typical series of spectra which demonstrate a loss of reflectivity and a shift in peak wavelength as a function of time and trypsin concentration in the cuvette. Figure 3 also shows a symmetric broadening of the peaks which is symptomatic of fringe loss. Trypsin concentrations in the assays were chosen to be nominally 25, 50, 100,200,350, and 500 nM, based on the trypsin being a pure dry protein with a molecular mass of 23 800. In order
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Figure 3. Timed sequence, at 1 min intervals, of spectral responses from a reflection hologram showing the changes in peak wavelength and reflectivity due to the action of 200 nM trypsin on the holographic medium. Sequential peaks are indicated by a progressive reduction in reflectivity and shift of the wavelength peak to the right.
to check for stability and to normalize different stock solutions from different days, trypsin activity was also measured by a spectrophotometric assay at 25 “C using sodium benzoyl-DLarginiine-pnitroanilide, (BAPN& Sigma B-4875) as substrate. For each assay, 20 pL of BAPNA (44 mg/mL in DMSO) was added to 0.97 mL of 12.5mM borax/HBr, pH 8.1. The reaction was initiated by addition of 10 pL of trypsin solution, and absorption at 405 nm was recorded for about 5 min. Activities were calculated by assuming an extinction coefficient of 1.02 x 104 M-’ cm-I for product and gave values of 0.62, 1.25, 2.21, 4.56, 7.59, and 11.28 nmol of product/min for the above nominal concentrations in 1 mL of solution.
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time (minutes) Flgure 4. Change in peak wavelength response to trypsin over time at activities of 0.62, 1.25, 2.21, 4.56, 7.59, and 11.28 nmol (trypsinBAPNA product)/min, seen from right to left on the graph.
RESULTS AND DISCUSSION
Response Curves. Data were recorded from a set of holograms designed to replay withiin a narrow range (665-693 nm) of stabilized peak wavelengths. Peak wavelength and reflectivity were monitored as a function of time until, at higher concentrations of trypsin, the peak reduced into the baseline ripple after about 20 min. Temporal response, peak wavelength change, and the standard deviation over four holograms per trypsin concentration are shown in Figure 4 and the corresponding calibration curves in Figure 6. Figures 4 and 6 show that discrimination of trypsin activity depends on the time elapsed and on the time window for observation. For peak wavelength measurement, it is clear that about 15 min must elapse before a useful range of changes is observed, while above 20 min, peak loss militates against quantitative evaluation of the holographic changes. Temporal response, peak reflectivity change, and standard deviation for four holograms per trypsin concentration are shown in Figure 5 and the corresponding calibration curves in Figure 7. Three features are evident from inspection of these data: first, an early slight rise in reflectivity, which is always seen at lower trypsin levels but which is dominated by intensity loss at higher trypsin levels; second, a flattening of the profile to a common level of change (about -80%) is usually observed; while the most significant feature is the large response recorded even for low levels of trypsin after 10 min. These data coni3n-1that the useful time window is 10-15 min for discrimination of trypsin activity. The zero concentration control ‘‘curves”are not shown because no change or drift from the zero baseline was observed outside the resolution capabilities of the instrumentation, f0.1% wave length change.
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time (minutes) Figure 5. Change in peak reflectivity response to trypsin over time at activities of 0.62, 1.25, 2.21, 4.56, 7.59, and 11.28 nmol (trypsinBAPNA product)/min, seen from right to left on the graph.
Mechanism of the Response. The extent of the responses described in this work is believed to be dependent not only on the nature of the matrix or gel that holds the hologram and its chemical composition and structural strength but also on the dependence of refractive index on structure. The shift in peak wavelength is caused by swelling of the gelatin matrix in aqueous solution and the consequent increase in separation of the fringes. Swelling is attributed to a weakening of the gelatin network by proteolytic cleavage of peptide bonds by trypsin. Swelling will continue until internal electrostatic, osmotic, and viscoelastic forces balance each other. In the case of a protease-catalyzed reaction, weakening of the gel is progressive and equilibrium is not reached as long as the protease is active and cleavable sites are available. The large reduction in peak reflection efficiency that occurs after the initial swelling can be attributed partly to disordering of fringes as the gelatin structure is degraded and partly to loss of Analytical Chemistry, Vol. 67, No. 23, December 1, 1995
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activity of trypsin (nmol produdmin) Calibration curves for peak wavelength response to trypsin activity, at 10, 15, and 20 min, seen from bottom to top on the graph. Figure 6.
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activity of trypsin (nmol producffmin) Figure 7. Calibration curves for peak reflectivity response to trypsin activity, at 10, 15, and 20 min, seen from top to bottom on the graph. fringecontaining gelatin. Disordering of fringes is likely to occur as the gelatin matrix containing silver bromide reorganizes following cleavage of nearby bonds. Removal of fringes is most likely because a broadening of the peak has also been observed (Figure 3): It is well-known within Bragg optics that fewer fringes contributing to constructive interference results in a broader band spectral peak, and a mathematical model of spectra has shown that this is the case. Loss of gelatin from the hologram has been confirmed by use of an acoustic wave sensor, sensitive to mass loss, where trypsin action on a gelatin overlayer caused removal of gelatin into solution. After several hours of exposure of the Agfa film to the reported levels of trypsin, the gelatin layer was completely removed from its mechanical substrate. A plausible hypothesis as to why the change in reflectivity levels off before attaining a change of -100% could be that the net biphasic nature of the gelatin hologram, i.e., alternate layers of silver bromide gelatin and gelatin alone, causes two rates of 4232
Analytical Chemistty, Vol. 67, No. 23, December 1, 1995
cleavage, whence the plateau on the temporal curve of Figure 5 would be followed by a further decrease in reflectivity toward -100% change. This was seen in a separate experiment, not presented here, where the spectral peak remained well-defined and measurable over the complete time course of the degradation. Note that the wavelength changes of Figure 4 do not level off at the same time as the reflectivity change but proceed because the weaker matrix continues to swell until reaching equilibrium. The initial rise in reflectivity at lower concentrations of trypsin is thought to be due to an increase in the depth of modulation of the refractive index produced when trypsin cleaves preferentially at sites between the fringes of silver bromide-containing gelatin. This may arise because the enzyme penetrates to these "empty" regions faster and/or its rate of cleavage may be slower at sites on gelatin immobilized around silver bromide grains. Fringe removal is likely to dominate as time progresses. Potential for Specificity. The multiple components contained within a real sample are each likely to have their own effect on a holographic element such as the one described above. In the detection of depressed pancreatic trypsin levels, for example, the sample will not only include other proteases such as chymotrypsin but also hydrolases such as lipase and amylase. Incubation of the holographic element with lipase and amylase produced no change in the reflected spectrum. However, the gelatin holographic substrate is expected to confer general specificity for proteases. Trypsin will cleave bonds on the carboxyl side of arginine and lysine residues in gelatin while other enzymes, chymotrypsin for example, also present in pancreatic fluid and therefore a possible contaminant of commercial trypsin preparations, can produce the same effect on a hologram as trypsin but by cleaving peptide bonds at leucine and phenylalanine residues. In a separate control, a chymotrypsin inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone (lT'CK) was used to treat the trypsin preparation as a safeguard against such contamination. However, no difference in response was observed between treated and untreated trypsin. In a second control, inhibition of trypsin by sodium #-tosyl-L-lysine chloromethyl ketone (TLCK) gave no holographic response, thus providing further evidence that the data presented above are due to trypsin alone. In the presence of potential interferents, pretreatment with TLCK would constitute part of a reference test. An alternative means of providing a reagentless reference test for interferents comprises blocking or modifying selected residues such that the susceptible cleavage sites for a particular enzyme are no longer available. For example, in the case of trypsin, arginine and lysine residues could be chemically modified in order to provide a trypsin-insensitive reference strip. Improvement and extension of the technique to obtain specsc responses to other enzymes is expected to utilize holographic structures made in non-gelatin materials which contain the substrate for the enzyme in question, the substrate molecules, or a reaction product contributing to the overall structure and stability of the material. Holographic tests could be envisaged for most hydrolytic enzymes. Practical Embodiments. The tests were carried out using a laboratory-based reflection spectrometer. A standard transmission spectrometer commonly found in research and clinical laboratories will suffice to measure hologram response whereby the absorption mode will correspond to reflectance. Departing from the large instrument scenario, the holographic element itself,
mounted on a suitable mechanical substrate, is a self-contained sensor in that a change in color or reflectivity can constitute a visually observable response. In order to achieve such a response to low trypsin levels, especially low fecal levels described above, it is apparent that a customized, possibly less cross-linked gelatin, must be used. In addition, whereas the above tests were done with holograms which replayed in the red, those to be monitored visually would preferably replay in the green to yellow region of the spectrum where normal eye response and color discrimination is good. Alternatively, if the illumination is by narrow-band light such as that obtained from a lightemitting diode, then response is seen as the changing brightness of the reflected light The data presented above show how rapidly the reflectivity of such a
device will change at low analyte concentrations, even when using a very hard holographic material. ACKNOWLEDGMENT The authors acknowledge the Biotechnology and Biological Sciences Research Council and The Leverhulme Trust for hancial support. We also acknowledge advice from our colleagues in the Institute of Biotechnology. Received for review August September 20,19 9 5 . ~
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Accepted
AC950868L Abstract published in Advance ACS Abstracts, October 15, 1995.
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