32 - Single-Cell Measurements of Purine Release Using a Micromachined Electroanalytical Sensor

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Single-Cell Measurements of Purine Release Usinga Microma chined Elec troanalytical Sensor

Craig D. T. Bratten,†,‡ Peter H. Cobbold,‡ and Jonathan M. Cooper* ,†

Bioelectronics Research Centre, Department of Electronics and Electrical Engineering, University of Glasgow, G12 8QQ,U.K., and Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool, L69 3EG, U.K.

To study the cellular events surrounding the formation of

purines in cardiac ischemia, we have micromachined a

micrometer-scale titer chamber containing an integrated

electrochemical se nsor, c apable o f measuring analytes

produce d by a single heart cell. The analytical proce dure

involves the determination of metabolites via the ampero-

metric detection of enzymically generated hydrogen per-

oxide, measured at a platinized microelectrode, poised

at a suitably oxidizing potential, equivalent to+

4 2 0 m Vvs Ag|AgCl. Signals were recorded as current-time

response s and were integrated to give a total charge (Q )

attributable to the reaction under investigation. The

amount of analyte produced by the c ell was s ubseque ntly

quantified by the addition of a known amount of calibrant.

As a conse quence, by using a cascade of three enzymes

(adenosine deaminase, nucleotide phosphorylase, and

xanthine oxidase), we were able to show that, after rigor

contracture had been induced in a single myocyte, ad-

enosine (but not inosine) only reached the extracellular

space after the cell m embrane had been permeabilized

by detergent. Thes e data, which could only be obtained

unambiguously by using this single-cell methodology,

have provided us with information on the origin of

ischemic adenosine which challenges the established

assumption that purine release is an early retaliatory

response from intact anoxic myocytes .

The study of single cells has generated data which could not

have been obtained using traditional in vitro methodologies. For

example, the measurem ents of cytoplasmic Ca2+ and ATP in s ingle

isolated cardiomyocytes have previously revealed new temporal

relationships during metabolic inhibition which were not apparent

from studies involving cell populations or the whole heart.1-4 In

the understanding of myocardial physiology, there is a consider-

able interes t in the role of the release of adenosine, a vasodilatory

catabolite of ATP5-7 which is believed to impair cell recovery upon

reoxygenation b y re ducing th e availability of intracellular purine

for the resynthesis of ATP. To investigate more closely the

processes governing the release of purines during cardiac is-

chemia, we have developed a single-cell measurement technology,

consisting of an integrated three-electrode microsensor system,

microfabricated within a surface micromachined measurement

chamber. The reaction volume inside the device is ∼600 pL,

defining a titer chamber for the measurement of femtomolequantities of the purines (adenosine and inosine) generated by

single rat cardiomyocytes.

Previously established single-cell measurements in biomedi-

cine, including both patch clamp and fluorescence techniques,

have been used for the measurement of ion flux through

membr anes. More recently, electrochemical techniques have also

been developed for studying single cells using carbon fiber

microelectrodes.8-10 In one such case,10 these techniques have

been used to complement traditional single-cell fluorescence

measurements, providing elegant and corroborative data which

can provide “downstream” information on metabolic processes,

resulting from changes in ion transport across membranes.

Against this background,1-10 many of the arguments both for

and against the development of a new single-cell measurement

technology involving micromachined sensors have already been

established. For example, single-cell analysis has already shown

that it has the potential to deconvolute complex patterns of

messenger production with a knowledge of the cell’s history,

providing the investigator with a high degree of confidence as to

the quantitative nature of the dose-response characteristics.

Against this, it can be argued that single cells are less relevant as

in vitro models, as they are not influenced by their neighbors,

and because they are exposed to greater amounts of (substrate)

surface area and/ or solvent.

Currently, there is an interest among pharmaceutical compa-

nies in developing micromachined de vices, not least becaus e the y

offer the prospect of obtaining a unique insight into the interac-

t io ns b etwe en c ell s ig na ls in a m an ne r wh ic h is n ot† University of Glasgow.‡ University of Liverpool.

(1) Cobbold, P. H.; Bourne, P. K. Nature 1984 , 312, 444-446.

(2) Allshire, A.; Piper, H. M.; Cuthbertson, K.S. B.; Cobbold, P. H. Biochem. J.

1987 , 244 , 381-385.

(3) Bowers, K. C.; Allshire, A. P.; Cobbold, P. H. J. Mol. Cell. Cardiol. 1992 ,

24 , 213-218.

(4) Allue, I.; Gandelman, O.; Dementieva, E.; Ugarova, N.; Cobbold, P. H.

Biochem. J. 1996 , 319 , 463-469.

(5) Schrader, J.; Haddy, F. J.; Gerlach, E. Eur. J. Physiol. 1977 , 369 , 1-6.

(6) Berne, R. M. Circ. Res. 1980 , 47 , 807-813.

(7) Jennings, R. B.; Steenbergen, C. Annu. Rev. Physiol. 1985 , 47 , 727-749.

(8) Paras, C. D.; Kennedy, R. T. Electroanalysis 1997 , 9, 203-208.

(9) Swanek, F. D.; Chen, G. Y.; Ewing, A. G. Anal. Chem. 1996 , 68 , 3912-

3916.

(10) Finnegan, J. M.; Wightman, R. M. J. Biol. Chem. 1995 , 270 , 5353-5359.

Anal. Chem. 1998, 70, 1164-1170

1164 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998 S0003-2700(97)00982-7 CCC: $15.00 © 1998 American Chemical SocietyPublished on Web 02/18/1998



possible when neighboring cells are influencing the local environ-

ment. Importantly, in the case of the study of hear t cells in vitro,

such local interactions m ay include the r elease of chemical species

due to mech anical stresses between asynchronously contracting

cells.

One potential problem which may be associated with using

low volumes of cell media, as is the case in these experiments, is

that, with time, the concentration of metabolites may increase

above physiological levels. However, in the context of the

relatively short periods over which the experiments described in

this paper were conducted, it was considered unlikely that this

would significantly affect the res ponses of the cell. Notwithstand-

ing this, by us ing microfabrication and micromachining technolo-

gies to reduce the analysis volume, the device that we have

developed has a number of advantages over traditional methods

for studying pathoph ysiologies involving either cell culture or

whole tissue technologies. In particular, because the diffusion

lengths within our device are reduced (i.e., the distance between

the cell and the sensor is small), responses become fast and no

analyte is lost to bulk solution (with the flux of m etabolites,

produced by the cell, rem aining high). One important innovation

has involved the de velopment of a microsystem with a transparentglass base, which has provided us with a means to observe the

cell simultaneously by light microscopy during the electroana-

lytical measurement, thus allowing defined metabolic events to

be related directly to observed cellular changes. In the case of

the single myocyte used in this experiment, this is of particular

relevance, as the cell undergoes significant physical changes in

its appearance, which can be correlated to biochemical/ patho-

physiological changes during ischemia.

MATERIALS AND METHODS

Micromachining of Devices. Devices were micromachined

using photolithographic techniques, adapted from the semicon-

ductor industry and described by us in a previous publication.11

In brief, photocurable polyimide was spin-coated onto a planar

fabricated Au microelectrode array and used to define the

micrometer -scale wells with a diamete r of 200 µm and a depth of

20 µm (se e Figure 2). After fabrication, platinum was electrode-

posited onto the gold microelectrodes, providing a stable elec-

trochem ical surface for the low-overpotential detection of H2O2.12

Sensors were used either in a standard three electrode configu-

ration, with a silver|silverhalide reference,11 or b y u s in g a

combined reference and counter electrode, acting as a pseudo-

reference e lectrode.

Determination of P urines. For the determ ination of purines,a mixture of adenosine deam inase (AD), nucleoside phosphorylase

(NP), and xanthine oxidase (XOD) was microinjected into the

device th rough a thin film of mineral oil in order to prevent the

medium from evaporating. In the presence of adenosine, there

was an enzymic production of a stoichiometric quantity of H 2O2

from adenosine (eqs 1-4), which was subsequently measured

amperometrically, at the platinized working e lectrode (eq 5),

poised at +420 mV vs Ag|AgCl. These enzymes catalyzed the

following reaction cascade:

Data were recorded using a low-current potentiostat with in-house

PC-based data acquisition. Responses of the device were mea-

sured upon addition of adenosine to an optimized enzyme mixture

containing 150 units mL-1 adenosine deaminase, 150 units mL-1

nucleotide phosphorylase, and 75 units mL-1 xanthine oxidase in

10 mM HEPES-buffered Ringer’s solution, pH 7.4. The am ount

of the purine present was estimated from the total charge passed

(measur ed by integration of the i-t response), as shown in Figure

1.

Single-Cell Measu rements . Single cell measurements were

made in HEPES-buffered Ringer’s solution (125 mM NaCl, 2.6

mM KCl, 1.2 mM KH2PO 4, 1.2 mM MgSO 4, 1 mM CaCl2, 10 mM

HEPES; pH 7.4) covered with paraffin oil and mounted onto a

heated Faraday stage (37 °C). The device was viewed under a

stereomicroscope (MZ6, Leica, UK, at ×100). Individual myo-

cytes, isolated by collagenase perfusion,13 were selected by

micropipet, placed into the droplet, and allowed to settle into the

chamber. The enzymes were prepared as a stock solution, in the

same buffer, containing 10 mg mL-1 fatty acid-free bovine seru m

albumin. To compare results, all responses were normalized

against a calibrant containing 70 fmol of adenosine injected at th e

end of each measurement (e.g., see Figures 3-6).

As a model for anoxia, the single cell was poisoned by

microinjecting 10 mM 2-deoxy-D-glucose (2-DOG) and 10 µM

carbonylcyanide p-(trifluorometh oxy)phenylhydrazone (FCCP)

into the chamber. In many cases, at the end of the experiment,

the cell membrane was permeabilized by the addition of the

detergent saponin (∼50 m g m L-1) into the microanalytical

chamber to measure the cell’s contents.

Reagents. Reagent grade hydrochloric acid, chlorobenzene,

diethyl ether , acetic acid, and acetone were from Aldrich (Gill-

ingham, UK). The enzymes adenosine deaminase, nucleotide

phosphorylase, and xanthine were used as supplied from Sigma

(Poole, UK). Saponin, 2-deoxy-D-glucose (2-DOG), carbonyl

cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), adenosine,

inosine, lithium perchlorate, sodium thiosulfate, glacial acetic acid,and Decon 90 were also from Sigma. Probimide 7020 photopat-

ternable polyimide and OCG developer were from OCG (UK).

1400-31 photoresist was from Sh ipley (UK). Metals, including Au,

Cr, Ni, Ti, and Pd, were from Goodfellows (Cambridge, UK). All

chemicals were used as received.

RESULTS AND DISCUSSIONMeasurement of Adenosine. Using the stated ratios of

adenosine deaminase, nucleotide phosphorylase, and xanthine(11) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1997 , 69 ,

253-258.

(12) Johnson, K. W.; Bryanpoole, N.; Mastrototaro, J. J. Electroanalysis 1994 ,

6 , 321-326.

(13) Powell, T.; Twist, V. W. Biochem. Biophys. Res. Commun. 1976 , 72 , 327-

333.

AD: adenosine f inosine + NH3 (1)

NP: inosine f hypoxanthine + ribose-1-phosphate (2)

XOD: hypoxanthinef xanthine + H2O2 (3)

XOD: xanthine f uric acid + H2O2 (4)

H2O2 f O2 + 2H++ 2e

-(5)

Analytical Chemistry, Vol. 70, No. 6, March 15, 1998 1165



oxidase within the enzyme cascade, described above, the micro-

analytical device gave re sponses (Figure 1a) which produced cali-

bration curves (Figure 1b) showing good linearity up to concen-

trations of ∼220 µM (∼130 fmol of adenosine), an amount which

is more than the maximum predicted purine generation from a

single myocyte.14 The precision of these measurements was

limited by two major sources: errors due either to pipetting or to

the sur face (electrochemical) condition of the electrodes. The

standard error was estimated as (7.9%by comparing a geometrical

quantification of the amount of the aliquot injected (measuredunder a microscope) with a series of successive electrochemical

titrations of hydrogen peroxide, made using the same microstruc-

tured devices.

The detection limit for adenosine was determined as 0.2 µM

(measur ed as 2 SD above baseline noise), equivalent to ∼120 amol

of analyte. The se lectivity for the measu reme nt of adenosine was

confirmed by measuring the responses to the addition of an

equivalent amount (70 fmol) of potential interferen ces, including

ATP, ADP, AMP, and IMP (Figure 1c). The broadness of the

i-t curves, characteristic of the measure ment of adenosine, shown

in Figure 1a, was in contrast with the temporally shorter, and

hence sharper, “spike” responses which res ulted from the addition

of a number of potential interferences (Figure 1c) and which may(in part) be attributable to perturbations caused to the electrode

double layer, due to th e action of pipetting. Additional controls,

performed in the pres ence of cells, were also carried out to exclude

the possibility of intracellular electroactive species contributing

to the response (see below).

Calibration of Respons es . Microanalytical chambers which

had been fabricated with platinized electrodes proved to give more

reproducible results than those with evaporated gold electrodes,

although inevitably ther e was intrade vice variability, attributable

to the history of the electrochemical surface (including, for

example, the electrode’s age, the amount of protein adsorbed, and

the electr ochem ical potentials previously applied) . Replatinization

of the electrodes was used to regenerate the electrochemical

interface, but this process also increased the surface area of the

device, hence producing variability in both signal and background

currents.

To quantify biological measu rem ents, pr ior to each single-cell

analysis, the device being used was calibrated using a series of

standard additions of adenosine (a representative example of this

process is shown in Figure 1a and b). After cell measurements

had been made, the experimentally measured results were

quantified by determining the charge generated as a consequence

of the addition of a known calibrant, containing 70 fmol of

adenosine. By using this method, imprecision could be attributed

either to nonlinearity of the sensor over the relevant dynamic

range of analyte (in which case the device could be discarded

prior to m easurement) or to substantial changes to the character

of electrochemical surface during the cell measurement (which

were minimized due to the r elatively short m easurem ent periods).

Single-Cell Measurements. The rat cardiomyocyte was

placed into the measurement chamber using a micropipet and

was manipulated manually into a central position (Figure 2a). As

a model for anoxia, the cell was metabolically inhibited by

microinjecting 10 mM 2-DOG and 10 µm FCCP into the microme-

(14) Allen, D. G.; Orchard, C. H. Circ. Res. 1987 , 60, 153-168.

b

a

c

Figure 1. (a) Representative responses of the device to additionof adenosine to 10 mM HEPES-buffered Ringer’s solution, pH 7.4:

[1] injection of ∼70 pL of buffer as control for other addition artifacts;[2-4] giving final concentration of adenosine as 1.66, 13.3, and 110µM, respectively. H2O2, produced enzymically (eqs 1-3), wasdetected electrochemically according to eq 4, as described in the text.

(b) An example of a calibration curve showing a linear response upto 130 fmol (∼220 µM) of adenosine. Responses remained linear over

the time course of a cell experiment (r ) 0.98). (c) Trace displayingselectivity of detection protocol: [1] injection of AD/NP/XOD to 150,

150, and 75 units mL-1, and then ∼70-pL additions of 1 mM IMP [2],AMP [3], ADP [4], ATP [5], adenosine [6], and inosine [7].

1166 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998



ter-scale cham ber. After a variable length of time, the poisoned

cell shortened to between 75 and 50%of its original length, but

still maintained its rod-shaped morphology, in a rigor-mediated

contracture process (Figure 2b). The cell membrane integrity

could be breached by permeabilization with saponin, after which

time the signal due to the generated adenosine could be measured

(Figure 3a). In all cases, the current-time (i-t ) responses,generated by formation of purines by the cell, were integrated to

give the charge (Q), which was quantified by direct comparison

with th e integr al of a signal from the addition of 70 fmol of purine

calibrant.

Control experiments on unpoisoned, rod-shaped cells were

performed by permeabilizing the membrane with saponin (Figure

3b). This caused the cell to round-up immediately, as shown in

Figure 2c, and showed that a significantly reduced amount of

adenosine was released from the unpoisoned cell than was the

case for the poisoned cell (Figure 3a) and Table 1. It was also

noted that both of the adenosine responses (Figure 3a,b), had

higher peak currents and were broader than the “spike” re-

sponses, recorded on addition of potential interferences (Figure

1c), including the response from addition of the detergent saponin,

shown in Figure 4a. Although some control responses had

significant peak currents, their absolute coulometric values were

small compared with the experimental measurements.

An additional control using poisoned, contracted single cells

(Figure 4b), permeabilized in the absence of all three enzymes

(AD, NP, and XOD), did not result in an electrochemical response.

This result confirmed that other electroactive species, including

intracellular ascorbate, as well as any other species which may

be present as a consequences of metabolic inhibition, such as

Figure 2. (a) Use of the nanovolumetric devices to investigate

purine generation by a single myocyte. The figure shows a view ofthe circular platinized working electrode, situated at the bottom of

the microelectrochemical chamber (diameter, 200 µm), and encircledby a (larger) counter electrode and a reference (see ref 11 for details).

A myocyte was inserted into the chamber using a blunted micropipetand oriented centrally in device. The medium surrounding the cell

was then injected with AD/NP/XOD to measure adenosine and inosinegeneration, or with NP/XOD to measure inosine only. (b) Typical

appearance of a cell after rigor contracture, induced by injectingmedium containing metabolic inhibitors (see text for details). Addition

of saponin (to release the intracellular contents) after rigor did notinduce any change in the cell shape. (c) Saponin permeabilization of

unpoisoned, rod-shaped cells resulted in their immediate “rounding-up”, probably as a result of large amounts of Ca 2+ entry.

F igu re 3 . Current-time (i -t ) traces showing myocyte purine

generation. (a) Adenosine generation from the myocyte following rigorcontracture is only measurable after saponin (∼50 mg mL-1) has been

injected into the microwell, to permeabilize the cell membrane. Thetrace also shows the addition of a calibrant of adenosine (70 fmol),

which is used directly to quantify the size of the recorded coulometricresponse. (b) Adenosine generation of healthy, rod-shaped cell, after

being permeabilized with saponin (∼50 mg mL-1). Again, the traceshows the addition of 70 fmol of the adenosine calibrant.




intracellular acidosis or buildup of NADH, did not interfere with

the electrochemical measurement.

Inosine gene ration was measured using the sam e protocol as

above (Figure 5a,b), although adenosine deam inase (but not NP

and XOD) was omitted from the enzyme cascade, such that

detection was via eqs 2-5 only. Data obtained showed that a

greatly reduced amount of inosine was generated during the

process of cellular metabolic inhibition, (Table 1, (i) and (ii)),

either by a healthy cell (Figure 5b) or by a cell after rigor

contracture (Figure 5a). This result indicated that the AMP

deamination pathway to IMP and subsequent inosine generation

by 5′-nucleotidase was not active under the conditions for cell

measurement, a finding which is consistent with the belief that

most purine catabolism to inosine takes place in the heart

endothelia and not within the myocardial cells.15,16

Adenosine generation by isolated healthy, rod-shaped cells,

when permeabilized by saponin, is considerably higher (38 ( 10

fmol, n ) 8) than that predicted (2.6 fmol) from whole tissue

studies15,17 (Table 1, (iii)). Given the cons trained volume in which

cells were permeabilized, we could not discount a significantamount of purine generation, either by membrane-bound ecto-

enzymes16 or by re leased cytosolic enzymes.18 Furthe r, we would

have expected that per meabilization in the presen ce of millimolar

concentr ation of extracellular Ca2+ will lead to a maximal activation

of myosin ATPase and may induce a catastrophic degradation of

ATP ( via ADP) to adenosine. Evidence supporting this was

obtained when cell permeabilization was per formed in a Ca2+-free

medium, achieved by adding both saponin and ethylene glycol

bis(β-aminoethyl ether) tetraacetic acid (EGTA) at a concentration

of 10 mM. Under these conditions, there was no immediate

shortening of the cell, but a Ca2+-independent rigor contracture

occurred after between 30 s and 1 min, probably as a result of

falling ATP levels.14 This was accompanied by a coincident startof a rise in the aden osine signal (69 fmol( 8.1, n ) 4, e.g., Figure

6).

In all experiments, there is a large variation in the time between

initial m etabolic inhibition and the onset of rigor contracture,19

which makes the estimation of adenosine formation during this

time difficult. In two cases, however, the ce ll was perm eabilized

precisely at the point of rigor contracture. The me an adenosine

produced (83 fmol) suggests a relatively small increase in(15) Rubio, V. F.; Wiedmeier, T.; Berne, R. M. Am . J. Physiol. 1972 , 222 , 550-

555.

(16) Meghji, P.; Pearson J. D.; Slakey, L. L. Am. J. Physiol. 1992 , 263 , H40-

H47.

(17) Frangakis, C. J.; Bahl, J. J.; McDaniel, H.; Bressler, R. Life Sci. 1980 , 27 ,

815-825.

Table 1. Purine Generation by Single Myocytes

experimentmean purine

gener ation/ fmol

(i) inosine generation byunpoisoned rod-shaped cell

3.8 ( 0.8 (n ) 4)

(ii) inosine ge neration bypoisoned cell after r igor contracture

7.5 ( 2.5 (n ) 4)

(iii) adenosine generation byunpoisoned rod-shaped cell

38.0 ( 10 ( n ) 8)

(iv) adenosine generation bypoisoned cell before rigor contracture

50.1 ( 15 ( n ) 4)

(v) adenosine generation bypoisoned cell after r igor contracture

160.3 ( 29 ( n ) 11)

Figure 4. Current-time (i -t ) traces showing myocyte purinegeneration. (a) Control measurement for the additin of saponin to a

normal rod-shaped cell in the absence of all three enzymes (AD, NP,

and XOD), indicating that the responses in Figure 3 are not due tointerference from the detergent, nor from the release of electroactive

species from the cytoplasm. (b) A comparable control for a poisonedcontracted single cell, permeabilized by saponin in the absence of

all three enzymes (AD, NP, and XOD), which again did not result in

an electrochemical response due to interference from the cell’scontents.

F igu re 5 . Current-time (i -t ) traces showing myocyte purinegeneration. (a) Response of a poisoned myocyte after cell perme-

abilization by saponin, made in the presence of the enzymes NP andXOD (but not AD). The addition of a standard containing 70 fmol of

inosine demonstrates that cellular inosine would have been measured,if present. (b) A representative trace from a control experiment

showing the lack of inosine generationg by a healthy myocytepermeabilized with saponin (∼50 mg mL-1). Again, the addition of a

standard containing 70 fmol of inosine demonstrated that cellularinosine would have been measured, if present.




adenosine formation, when viewed in the context of the data for

pre-rigor conte nt ( 50 fmol ( 15, n ) 4, Table 1, (iv)). In contrast,

the generation of adenosine after rigor contracture was consider-

able, with the mean (160 fmol ( 29, n ) 11, Table 1, (v))

correlating closely with estimates of ATP content in normal

cells.14,20 In a minority of these experiments (n ) 2), the

membrane of a poisoned cell ruptured spontaneously, and a large

adenosine response (mean 116 fmol) was obtained precisely

coincident with contracture ; addition of saponin did not yield any

further signal.

Thus, we have shown that the majority ofadenosine ge neration

occurs rapidly and accompanies rigor contracture, in an amount

which is equivalent to the cell’s ATP content. Despite the h igh

sensitivity of this device, no increase in baseline current was

detected from intact cells, even up to 10 min after rigor contracture

prior to permeabilization of the cell membrane, indicating unam-

biguously that the continuous release of adenosine from intact

cells is very slow. Over a similar time period, whole heart

perfusates already shows a significant rise in aden osine content.20-22

The amount of adenosine generated by the cell after rigor

contracture correlates well to values obtained from whole heart

studies, in which the tissue content of purine nucleosides h as been

shown to increase commensurably with the decline in ATP

levels.20,24 To date, the assumption has been that the purine

nucleosides, which appear in the coronary perfusate during cardiac

ischemia,18,24,25 are released from intact cells via the membrane

purine transporter.18,21,26,27 Here we show the unexpected and

striking observation that, for single cells, significant adenosine

only reaches the extracellular space after the cell has been lysed,

regard less of its metabolic condition. Not withstanding this latter

point, it should be emphasized that single-cell studies provide a

highly appropriate technology for conducting these investigations

into purine formation, as the myocyte is free of the mechanical

stresses between asynchronously (neighboring) contractions,

28

which may contribute to values observed for purine generation

from the whole he art. Indeed, we now speculate that a large part

of the adenosine release observed in whole heart or in vitro cell

population studies may originate from lysed cells.

The Nature of the Temporal Respons e. In analyzing the

temporal response of the single cells within the device, it is

important to consider carefully the many processes which are

occurring in the micromachined structure, and how these relate

to the measurements being made. These are illustrated schemati-

cally in Figure 7, which represents a myocyte contained within a

microanalytical electrochemical cell, in which h ydrogen peroxide,

oxygen, and the enzyme substrates all are in solution (with

diffusion coefficients , DH2O2, DO2, and DS, all of which are allsimilar). Both the cell and the enzyme XOD (within the oxidase

enzyme cascade) require oxygen, which must be partitioned

across the mineral oil (K O2) into the electrochemical cell. The

cell will obtain this oxygen, first by diffusion through the buffered

medium (DO2) and then by transfer across its plasma membrane

(K′O2). The fact that the cell can survive within the device for

periods up to 2 h (the maximum tested) would indicate that the

partitioning of oxygen into the chamber is greater than or equal

to the equivalent term for the partitioning of oxygen across into

the cell membrane (i.e., K O2) K ′O2

).

In alternative assay schem es, ther e is th e possibility that, when

the detection enzyme is an oxidase, it may compete with the cell

for the available oxygen. However, in the measur eme nts that wedescribe in this paper, adenosine is only present after cell lysis,

(18) Schutz, W.; Schrader, J.; Gerlach, E. Am . J. Physiol. 1981 , 240 , H963-

H970.

(19) Haworth, R. A.; Hunter, D. R.; Berkoff, H. A. Circ. Res. 1981 , 49 , 1119-

1128.

(20) Geisbuhler, T.; Altschuld, R. A.; Trewyn, R. W.; Ansel, A. Z.; Lamka, K.;

Brierley, G. P. Circ. Res. 1984 , 54, 536-546.

(21) Van Belle, H.; Goosens, F.; Wynants, J. Am. J. Physiol. 1987 , 252 , H886-

H893.

(22) Vanwylen, D. G. L.; Schmit, T. J.; Lasley, R. D.; Gingell, R. L.; Mentzer, R.

M. Am. J. Physiol. 1992 , 262 , H1934-H1938.

(23) Jennings, R. B.; Reimer, K. A.; Hill, M. L.; Mayer, S. E. Circ. Res. 1981 , 49,

892-900.

(24) Katori, M.; Berne, R. M. Circ. Res. 1966 , 19 , 420-425.

(25) Olsson, R. A. Circ. Res. 1997 , 26 , 301-306.

(26) He, M.-X.; Gorman, M. W.; Romig, G. D.; Meyer, R. A.; Sparks, H. V. Am .

J. Physiol. 1991 , 260 , H917-H926.

(27) Chen, W. N.; Hoerter , J.; Gueron, M. J. Mol. Cell. Cardiol. 1996 , 28, 2163-

2174.

(28) Siegmund, B.; Koop, A.; Klietz, T.; Schwartz, P.; Piper, H. M. Am. J. Physiol.

1990 , 258 , H285-H291.

(29) Cass, A. E. G.; Davis G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins,

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667-671.

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69 , 543-551.

Figure 6 . Delay of contracture of healthy cell due to saponininjection in Ca2+-free medium (achieved by addition of saponin with

10 mM EGTA), with the onset of adenosine generation coincidentwith rigor contracture.

Figure 7. Transverse section of a schematic diagram of the physicalprocesses occurring within a microelectroanalytical chamber contain-

ing a counter (C) and working (W) electrode, in which an enzymecascade (E) converts substrate (S) to product (P). See text for full

details.




and thus it can be assumed that the cell is not competing with

the enzyme for oxygen and that the de tection reaction is able to

go to completion. That the enzyme cascade can be used to

measure adenosine while the cell is still alive, as evidenced

through a standard addition of analyte, supports the general

conclusion of this paper, namely that adenosine is only released

from the myocyte after its plasma membrane is breached.

The advantage of using a chronocoulometric “end-point”

measurem ent, integrated over the whole time course of a reaction,

is that it is possible to quantify the purine signal, regardless of which of a number of processes is the rate-determining step (for

example, either the electrochemical oxidation rate constant for

the h ydrogen peroxide, k s, or the k cat for the enzyme cascade, E ,

may be rate determining, although in either case the experimental

result should be identical, as no analyte is lost to the bulk solution).

That this “end-point” is reached relatively quickly (Figures 3-5)

is due to the fact that the diffusion distances in the device are

short.

Despite the constraints imposed on the types of measurem ents

which can be made using these devices (see above), it should be

noted that this technology has a potential to further extend the

range of compounds that can be detected from single cells to

include the purine nucleosides. By incorporating different enzymesystems, these microsystems can be readily adapted to measure

a wider range of analytes from single cells, including glucose,

lactate, and glutamate. Although, in the future, there is the

possibility of adding a mediator29 to negate oxygen dependency,

it was felt that, at this stage, this would detract from the overall

simplicity of this detection method described.

Since the microelectroanalytical chambe rs are fabricated us ing

protocols adapted from standard photolithographic methods, there

is the potential to reduce the dimensions of the device further

and thus provide structures that are more appropriately sized for

smaller cell types, giving higher fluxes of analyte (perhaps

enabling faster response times). There is also the possibility of

having more than one sensor incorporated within each micro-

structure, to allow for detection of more than one analyte,simultaneously. And finally, in the longer term, we will produce

arrays of these devices and interface them with technologies for

the d ispens ation of low volume s of fluid; e.g., “ink-jet” tech nology

has already been used in order to develop h igh-throughput

secondary screens for drug discovery.30 Using su ch microfluidic

dispensations, there will be an ability to control more reproducibly

the position of the microcapillary tip when adding fluids and he nce

minimize (and q uantify) the n onFaradaic changes in th e electrode

double layer (seen as “spikes”) resulting from perturbances in

the medium during pipetting.

Acknowledgment is given to both the Wellcome Trust and

to Glaxo-Wellcome for supporting aspects of this research.

Received for review September 8, 1997. AcceptedJanuary 4, 1998.

AC970982Z


Documents

32 - Single-Cell Measurements of Purine Release Using a Micromachined Electroanalytical Sensor