A solid-phase optoelectronic sensor for serum albumin

ANAI.YTICAL

BIOCHEMISTRY

A Solid-Phase

109, 216-221

(1980)

Optoelectronic M.J.

Sensor AND C.R.

GOLDFINCH

Received

April

for Serum Albumin LOWE

4. 1980

A simple solid-phase optoelectronic sensor for serum albumin is described. Bromocresol green covalently attached to a cellophane membrane is sandwiched between a red light emitting diode and a silicon photodiode with integral amplifier. Adsorption of serum albumin to the membrane at pH 3.8 causes a characteristic yellow to blue-green color change in the membrane and is thus monitored as a fall in the output voltage of the detector system. The response is reproducible and linear over the range 5-35 mgiml albumin concentration. This report describes an investigation of this inexpensive and potentially reagentless albumin sensor.

It has been known for a very long time that the measurement of pH with certain indicators is liable to error in the presence of appreciable amounts of protein. This effect, the “protein error,” manifests itself as a color change in the indicator and is due to a protein-dye interaction which is greatest when the pH of the solution is on the acid side of the isoelectric point of the protein. A number of dyes, especially methyl orange, 2-(4’-hydroxyazobenzene)benzoic acid and bromocresol green (l-3) have been claimed to bind relatively specifically to serum albumin with the color change being proportional to the concentration of albumin present. The binding of bromocresol green is widely used in the estimation of albumin in clinical biochemistry (3). The color of an aqueous solution of bromocresol green buffered to pH 3.8 changes from yellow to green or blue in the presence of albumin. The bromocresol green assay appears to be less prone to competition at the dyebinding site by bilirubin or drugs and shows good correlation with electrophoresis in specimens with inverse albumin/globulin ratios (4). In the present report, the dye-binding properties of albumin are exploited to con0003.2697/80/180216-06$02.00/O Copyright All rIghI\

10 1980 hy Academic Prr\r. Inc of rrproduct~on m any form rerervrd

216

struct a simple and inexpensive solid-phase optoelectronic sensor for serum albumin. The dye, bromocresol green, has been covalently attached to a cellophane membrane and sandwiched between a red light emitting diode and a detector comprising a silicon photodiode with integral amplifier. This paper reports preliminary observations on the construction and response of the sensor. EXPERIMENTAL Materids. Bromocresol green. bovine serum albumin (fraction V; 96%), N-glycylglycine, 2-mercaptoethylammonium chloride, and the ninhydrin spray were purchased from BDH Chemicals Ltd., Poole. Dorset. England. Reduced glutathione (crystalline) and 1,6-diaminohexane were from the Sigma (London) Chemical Company, while cyanogen bromide (97%) was obtained from the Aldrich Chemical Company, Milwaukee, Wisconsin, and Sepharose 4B from Pharmacia (G.B.) Ltd. The thin-layer materials. DC Fertigfolien F1500 LS 254 Kieselgel, DC Fertigfolien Fl440 LS 254 cellulose, and DC Fertigfolien F1440/LS 254 PEI-Cellulose were from Schleicher & Schull. Dassal. West Germany. The cello-

AN ALBUMIN

SENSOR

217

(R, 0.71; DC Fertigfolien Fl500 LS 254 Kieselgel; n-butanol/acetic acid/water: 4: 1:5, v/v/v; upper phase), a mauve product (R, 0.58), and finally the yellow-green bromocresol green-glutathione conjugate (R,0.34). Unreacted glutathione remained adsorbed to the top of the silica column and gave a positive reaction to ninhydrin. The yellowgreen product (R, 0.34), recovered in approximately 10% overall yield, was concentrated by rotary evaporation at z 6O”C, Synthesis of the bromocresol green-gluredissolved in water, and repeatedly evapotclthione conjugate. It is known that a rated until all traces of acetic acid were sulfobromophthalein - glutathione con- removed. The yellow product which bejugate forms gradually when sulfobro- comes dark blue in water of pH -5.0, was mophthalein and reduced glutathione are finally lyophilized. The resultant purple incubated at pH 7-10 (5-7). Preliminary powder was homogeneous on thin-layer incubations of bromocresol green with chromatography (silica gel: R, 0.34; nthiols such as reduced glutathione and 2- butanoliacetic acid/water, 4:1:5 (v/v/v). upmercaptoethyl ammonium chloride at pH per phase) and ninhydrin positive. In 1 M 6.5- 10 produced analogous conjugates. NaOH, A,,, = 450 nm, 620 nm: eh, = 42.550 Contrary to the observations of Whelan liter.mol~‘~cm~’ at 620 nm. Bromocresol et al. [6] with sulfobromophthalein, higher green: pK,, = 4.7; bromocresol green-gluyields of the bromocresol green-glutathione tathione conjugate: pK,, = 5.1. determined conjugate were obtained under mildly acidic in 100 mM sodium acetate buffer pH 3.6conditions. 5.8 at 450 and 620nm. Bromocresol green (840 mg; 1.4 mmol) Immobili~rrtior~ of the bromocresd grcet~ and reduced glutathione ( 1840mg; 6.0 mmol) glutathionr conjugate. Exhaustively washed were dissolved in distilled water; the pH was Sepharose 4B (6 g wet wt in 10 ml water) adjusted to 6.5-6.6 (0.880 NH,OH) and or Visking tubing (3 g dry wt in 10 ml water) the final volume, adjusted to 90 ml. The was activated with CNBr (140-200 mg/g) mixture was incubated at 40°C for 48 h, for 30 min at I2-15°C and pH 10.8 + 0.2. degassed under reduced pressure and vig- The activated supports were thoroughly orous stirring for 1 h, and then rotary evapo- washed with distilled water and 0.1 M ice rated at z 60°C to dryness. The residue was cold NaHCO,-Na,CO, buffer, pH 9.3. prior redissolved in ,I-butanoliacetic acid/water to adding the bromocresol green-glutathione (4:1:5. v/v/v: upper phase; 20 ml) and ap- conjugate (62 mg, 89 pmoV3.5 g wet wt plied to a column (60 x 240mm) of silica gel activated Sepharose 4B or 80 mg. 114pmol/ (Kieselgel 60 PF,,,; E. Merck, Darmstadt, 3.0 g activated Visking tubing) to the W. Germany) equilibrated with n-butanoli CNBr-activated supports. The coupling mixacetic acid/water (4: 1:5. v/v/v: upper phase). ture was tumbled gently for 18h at 0-4°C Fractions (10 ml) were collected at a flow and unbound dye conjugate removed by rate of approximately 30 ml/h maintained exhaustive washing with water, 0.1 M HCI, with a hand bellows. The column sep- 8 M urea, 1%Triton X-100, and finally water. arated the mixture into three major bands The dyed support matrices were stored comprising unreacted bromocresol green moist at 0-4°C. The concentration of the dye immobilized I Abbreviation used: LED, light-emitting diode. on the membrane and Sepharose 4B was phane membrane was dialysis tubingVisking size 5-24132 in. from Medicell International Ltd., London. The light-emitting diodes (LED’; Siemens Type LD 52-C; high brightness, 12” l/2 viewing angle) were from A. Marshall Ltd.. London, United Kingdom, while the Silicon photodiode with integral amplifier (Type 308-067) and all other electronic components were purchased from R.S. Components Ltd., London, United Kingdom.

218

GOLDFINCH

AND

LOWE

determined by hydrolysis of a known weight of matrix in 1 M NaOH for 1 h at 35°C and measurement of the absorbance at 620 nm using the molar extinction coefficient, Ed = 42.550 liter.mol~‘.cm~’ at 620nm. Afjnity chromatogrrrphy of albumin. A column (1.0 X 3.0 cm) containing agaroseimmobilized bromocresol green (2 g wet wt) was equilibrated with 20 mM sodium citrate buffer pH 3.8 or 4.5 or 20 mM sodium phosphate buffer, pH 7.0. and bovine serum albumin (I ml: 2 mgiml) applied at 20°C. Nonadsorbed protein was washed off with starting buffer and elution effected with buffer containing 0.5 M KCl.

type 308-0671, were placed on either side of the flow cell and located in holes 5.1 and 8 mm diameter, respectively, drilled in two further sheets of lucite 5 mm thick. All four lucite sheets were accurately located with metal studs to ensure perfect alignment of the LED, membrane, and detector. and bolted together. The whole assembly was mounted vertically with the inlet hole in a ventral position and encased in an aluminumlight-proofbox, 12.4 x 7.5 x IOcm. A dual-output stabilized power supply unit (Type LT30Il; Fame11 Instruments Ltd., Wetherby, Yorks, U. K.) was used to power both the LED through an external series current-limiting resistor (470 a 11W) Construction of the optorlectronic senand the detector photodiode circuitry. The SOI’. The optoelectronic sensor comprised voltage across the LED was typically about a flow through cell, 4 mm deep and 8 mm 1.885V. The output voltage (V,,) of the diameter cut into a 5 mm-thick lucite sheet photodiode amplifier was monitored on a (50 x 50 mm) with 2-mm diameter feed Sinclair Model DM 450 4% digit multimeter and outlet holes and closed on the open (Sinclair Electronics Ltd., Cambridge. U. K.) side with a second lucite sheet 2 mm thick. across a 47K 0 2% load resistor. A circuit The bromocresol green membrane (IO x 10 diagram of the albumin sensor is given mm) was sandwiched between the two in Fig. 1. The emission spectrum of the red LED blocks. The light source, a red light-emitting diode (LED) (Siemens; high brightwas examined by carefully positioning the ness: 12” % viewing angle: Type LD 52-C) diode immediately in front of the photoand the detector, a silicon photodiode with multiplier/grating window of a Pye- Unicam integral amplifier (Radiospares Components SPl800 Spectrophotometer in single-beam t

FIG. I. Circuit Diagram for the solid-phase optoelectronic sensor for serum albumin; (a) lightemitting diode. red, high brightness, Siemens. Type LD52-C; (b) Silicon photodiode with integral amplifier. Radiospares Components Type 308-067: (c) dual output stabilized power supply: (d) circuit to provide c I5 from 30 V stabilized line: (e) membrane housed in kite flow-through cell: (f) aluminum light-proof box: tg) output voltage monitored on a Sinclair DM 450 digital multimeter.

AN

ALBUMIN

operation. The emission maximum, 630633 nm. was slightly lower than the published value of 645 nm. Sunlple ~IS.SU~ procrdurr . The output voltage (V,,) of the photodiode amplifier was initially set to 7.000 V by suitable adjustments to the stabilized power supply when 20 mM citrate buffer. pH 3.8, was pumped through the flow through cell at 4 mlimin. The pH response of the membrane-immobilized dye was measured by monitoring V,, as a function of pH using 100 mM acetate buffers. pH 3.3-5.x. and 100 rnhl phosphate buffers, pH 6.3 and 6.8. For the determination of albumin, V,, was set to 7.000 V in 30 mM citrate buffer, pH 3.8. the albumin sample in the same buffer pumped through, the new V,, recorded. 8 M urea pumped through. and finally the membrane reequilibrated with the citrate buffer, pH 3.8.

219

SENSOR 07-

06\

d

, 250

330

FIG. 2. Absorption glutathione conjugate pH 3.8. (al in the of serum

albumin

410

L90 h lnmi

570

650

7 730

spectraofthe bromocresolgreenin 20 mM sodium citrate buffer, absence, and (b) in the presence (2 mg/ml).

RESULTS The synthesis of the bromocresol greenglutathione conjugate and its subsequent immobilization was analogous to established procedures for bromosulfophthalein (6.7). The R, value on silica gel thin-layer chromatography, positive reaction to ninhydrin. ability to couple to CNBr-activated polysaccharides, absorption maxima, and molar extinction coefficient at 630 nm all concur with the formation of a covalent dyeglutathione conjugate. The modified dye in free solution displays a pK,, of 5.1. slightly higher than unmodified bromocresol green at 4.7. Furthermore, Fig. 2 demonstrates that like the unmodified dye the bromocresol green-glutathione conjugate binds serum albumin at pH 3.8 with a significant increase in absorbance at 620 nm and hence a color change from yellow to blue-green. This color change is also observed on binding serum albumin to agarose-immobilized bromocresol green. At pH 7.0 the applied albumin is quantitatively recovered in the void volume of a column containing

0.45 pmol dye/g gel. At pH 4.5, retardation equivalent to 2 column vol was observed while at pH 3.8 all the applied albumin (2 mg) was bound and subsequently eluted quantitatively with 0.5 M KC1 or 8 M urea in the same buffer. The color change observed on binding albumin to immobilized bromocresol green and detected at 620 nm forms the basis for the optoelectronic sensor (Fig. 1). The red LED emits light of A,,;,, 630-633 nm and generates about 60% of the maximum responsivity of the silicon photodiode amplifier at 900 nm. This coupled-light sourcedetector system is therefore ideally suited to monitoring albumin binding to bromocresol green membranes. Figure 3 demonstrates that the bromocresol green membrane responds to pH over the range 3.8-7.3 in the same way the free dye does with a pK,, -5.1. The color change of the membrane. containing 2.09 pmol dye/g dry wt. from yellow to bluegreen produces a 3 to 4 V change in output

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GOLDFINCH

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voltage from the photodiode. A conventional pH meter produces 300-400 mV over the same pH range. The standard curve for albumin in the concentration range O-45 mg/ml is shown in Fig. 4. The change in output voltage is approximately linear within the range 5-35 mg albumin/ml and equivalent to 13 mVlmg/ml increment in albumin concentration. At albumin concentrations greater than 35 mg/ml a progressive saturation effect is observed. The effects of pH and albumin on the membrane-bound dye and hence output voltage of the photodiode amplifier were completely reversible. After each albumin determination the membrane was regenerated with 8 M urea and the starting buffer, 20 mM citrate, pH 3.8. when the output voltage returned to its initial value of 7.000 V. In 10 separate measurement/regeneration cycles of the sensor with a standardized albumin concentration of 10.8 mg/ml the observed output voltage change was 164.20 IL 2.35 mV (SD). Each measurementiregeneration cycle was completed in approximately 8 min.

LOWE

DISCUSSION The binding of bromocresol green is widely used in the estimation of serum albumin in clinical laboratories. The technique appears to be less prone to competitive binding at dye sites by bilirubin, fatty acids. and drugs (4.7-9). Covalent attachment of bromocresol green to a thin cellophane membrane sandwiched between a light source. a red light-emitting diode (A,,,;,, emission 630-633 nm). and a detector, a silicon photodiode with integral amplifier, permits the characteristic color change from yellow to blue-green observed on binding albumin at pH 3.8 to be monitored. The technique is simple, inexpensive. and extremely reproducible. Furthermore, the method is potentially reagentless if the ambient pH is raised to effect retardation of the albumin rather than adsorption, as observed in the preliminary affinity chromatography experiments. This would obviate the need for a regeneration step of the membrane and thus permit serial measurements of albumin samples every few min-

70-

6 5-

60-T f2

55-

3 5-

30t

, 33

38

43

48

53

56

63

68

I 73

PH

FIG. 3. The effect of pH on the bromocresol ( I/,,) of the photodiode amplifier.

green

membrane

as monitored

by the output

voltage

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0

1 5

I 10

I 15

ALBUMIN

1 20

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SENSOR

I 2s [Albumin]

I 30

1 35

I 40

I 45

I 50

mg/ml

FIG. 4. The effect of albumin on the bromocresol green membrane as monitored by the output voltage (V,) of the photodiode amplifier. The ordinate is expressed in terms of the change in millivolts of the output voltage (V,) from its initial value of 7.000 V in the absence of albumin as a function of serum albumin concentration.

utes. Furthermore, a parallel array of severa1 LED/detector systems would allow proportionately greater numbers of samples to be processed per hour. The sensitivity of the sensor could be improved by using thinner cellophane membranes. It appears that albumin binds only to surface bound immobilized dye since the membrane response to albumin is markedly lower than the response to pH, presumably because protons can percolate throughout the whole membrane. The accuracy of the sensor appears to be limited primarily by the detector system since the output noise voltage of the silicon photodiode amplifier is typically 1-2 mV rms. Nevertheless, both the dye-membrane and the detector system are stable and entirely reproducible within these limits over a period of several months use. Indeed, the characteristics of the LED and photodiodeiamplifer are inherently stable for many thousands of hours. Thus, it is anticipated that with suitable minor constructional and procedural modifications the albumin optoelectronic sensor could be made reagentless, more sensitive, and ca-

pable of handling samples.

large numbers

of serial

ACKNOWLEDGMENTS This work Council.

is supported

by the

Medical

Research

REFERENCES I. Breyer. B.. and Bauer, H. H. (1953)A~sr. J. Chrm. 6, 332-339. 2. Peticolas. W.. and Klotz, I. M. (1956) .I. Amer. Chern. SW. 78. 3539-3542. 3. Kotes. J. (1966) CU.F. I,&. CCXA. C-r,sr 105, 790-795. 4. Morgenstern. S.. Rush, R.. and Lehman. D. ( 1973) in Advances in Automated Analysis. Vol. I. pp. 27-31. Mediad Inc., New York. 5. Jovitt. N. B.. Wheeler, H. 0. Baker. K. J.. Ramos, 0. L.. and Bradley. S. E. (1960) J. Clin. Invesr. 39. l570- 1578. 6. Whelan. G.. Hoch, J.. and Combes. B. (1970) J. Lab. Clin. Med. 75, 542-557. 7. Clark. A. G.. and Wong. S. T. (1979) Awl. Biothem. 92, 290-293. 8. Jacobsen, J.. Thieseen, H., and Brodersen. R. ( 1972) Biochrm. J. 126. 9. Travis, J.. Bowen. J.. Tewksbury. D., Johnson, D.. and Pannel. R. (1976) Bi~~~~/rrm. J. 157, 301-306.