DETECTION OF THE CYTOMEGALOVIRUS

A Mobile Device and a Disposable Cartridge for Detection at the Patient's Bed

Thomas Mangeat, Hichem Benalia, Christian Pieralli, Alain Rouleau, Wilfrid Boireau

Bruno Wacogne

FEMTO-ST Institute, UMR 6174, 16 Route de Gray, 25030 Besançon cedex, France

Jean-Sébastien Guerrini, Alain Coaquette, Georges Herbein, Lionel Pazart

Besançon University Hospital, 2 Place Saint Jacques, 25030 Besançon cedex, France

Christian Davrinche

INSERM U 563, Paul Sabatier University, Toulouse, France

Keywords: Biosensors, Embarked system, Cytomegalovirus, Immunofluorescence.

Abstract: Recently, cytomegalovirus (CMV) infection has become the most frequent cause of congenital infections.

The French Health Authority (HAS) is urging a diagnosis at birth for newborns. Since no screening device

is commercially available, a consortium has been established to set-up an original device. It consists of a

disposable cartridge containing the biological sample and the reactive liquids required for

immunofluorescencence detection on a functionalized surface. It also consists of a mobile reader used to

drive the fluids onto the biosensor and to ensure the optical measurement. Up to now, positive and negative

samples can be discriminated with a fluorescence intensity ratio of 3.

1 INTRODUCTION

Since the rubella vaccine was established, the

cytomegalovirus infection has become the most

frequent cause of congenital infections, particularly

in premature babies (prevalence between 2 and 10%

according to studies). Before deciding on the benefit

of screening in this population, the HAS (French

Health Authority) is urging "a study in newborns

(diagnosis at birth) with a long-term follow-up of

infected children” to be carried out. One of the

obstacles to carry out such a study lies in the

diagnostic means currently available (Demler-

Harrisson, 2009).

Congenital infections are the result of

transplacental transmission of CMV. Transmission

to the fetus may occur because of primary or

secondary maternal infection. The frequency of

intrauterine transmission following primary

infection during pregnancy is 30% to 40%,

compared with only 1% following secondary

infection (Stagno, 1986 – Raynor, 1993). Ten to

fifteen percent of congenitally infected infants will

have symptoms at birth, and 20% to 30% of them

will die (Raynor, 1993 – Nigro, 1999 – Pass, 2002).

Most of the congenitally infected infants (85–90%)

have no signs or symptoms at birth, but 5% to 15%

of them will develop sequelae such as sensorineural

hearing loss, delay of psychomotor development,

and visual impairment (Boppana, 1992 – Pultoo,

2000 – Lazzarotto, 2000).

Routine serologic screening for pregnant women

is rarely recommended by public health authorities

(Revello, 2002 – Yinon, 2010). The screening, if

done, should be performed at the beginning of

pregnancy or even prior to a planned pregnancy in

order to identify a seroconversion during pregnancy.

Instead of screening for pregnant women, some

authors recommend to screen babies at birth, but

reliable methods to screen newborns for congenital

cytomegalovirus (CMV) infection are still needed

(Fowler, 1999 – Boppana, 2010).

103

Mangeat T., Benalia H., Pieralli C., Rouleau A., Boireau W., Wacogne B., Guerrini J., Coaquette A., Herbein G., Pazart L. and Davrinche C..

DETECTION OF THE CYTOMEGALOVIRUS - A Mobile Device and a Disposable Cartridge for Detection at the Patient’s Bed.

DOI: 10.5220/0003120201030108

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2011), pages 103-108

ISBN: 978-989-8425-37-9

 2011 SCITEPRESS (Science and Technology Publications, Lda.)

The diagnosis of infection in newborns depends

on finding the virus in the different biological

liquids and more specifically urine which

concentrates the virus. Apart from CMV detection

kits for the laboratory, numerous lines of research

concern virus detection and Microsystems (µTAS,

MEMS, etc.) (Huikko, 2003 – Anderson, 2003).

These microsystems are generally dedicated to

detect genetic material after preparing samples, most

often by PCR or RT-PCR (Liao, 2005 – Park, 2004).

Concerning the biological fluid used and/or the type

of analysis, most examine blood cells or other types

of cells, which require virus extrusion operations of

the cells and a blood puncture for collecting

biological fluid. Indeed, a mobile device is required.

The microsystem presented here and developed

under the coordination of the FEMTO-ST Institute

in the framework of a 2006 ANR TecSan project,

approved by a microtechnics competitive cluster, is

an embedded detection device which uses a

microsystem including the functionalized surface for

CMV trapping (patent request submitted in

September 09). The detection and dosage of the viral

material are based on immunofluorescence

techniques, with materials and micromanufacturing

processes compatible with a low-cost industrial

production. Among the medical acts carried out at

birth, in particular in premature babies, gastric

aspiration allows a biological fluid combining foetal

urine (excreted as from the 5th month) with amniotic

fluid to be obtained easily. We therefore chose to

use this biological fluid as a screening medium.

In the next part of the paper, we describe the

immunofluorescence detection scheme as well as the

device we developed. The third part is devoted to the

bio-chemistry of the bio-sensor and to the first

experimental results concerning CMV detection.

Then a conclusion will be proposed to this work.

2 DESCRIPTION OF THE

DEVICE

The detection relies on the use of an

immunofluorescence biosensor depicted in figure 1.

The biosensor surface is coated with CMV specific

antibodies. A biological sample is then applied onto

the surface of the biosensor. If CMV is present in the

biological sample, it is trapped onto the surface by

means of the antibodies. Then, after washing the

surface with buffer, a fluorescent probe is injected.

The latter consists of complementary Cy5 labelled

antibodies. Therefore, if CMV is present in the

sample, a fluorescent signal is detected.

Figure 1: Immunofluorescence detection of CMV.

This biosensor is integrated into a disposable

cartridge (figure 2). It contains all the fluids required

for the immunofluorescence reaction. In the figure,

we can see the gold coated functionalized surface as

well as different deformable balloons. Four balloons

are used. One contains the biological sample to be

tested, a second one contains the fluorescent probe, a

third one contains the buffer and the last one is used

for waste. In this example, the biological sample is

injected into the disposable cartridge with a

conventional syringe. Micro-channels are used to

drive the fluids from the balloons to the

functionalized window where the reaction takes

place.

Driving the fluid and detecting the possible

fluorescence signal is performed into the mobile

device, hereafter the reader, shown in figure 3. The

disposable cartridge is inserted into the reader

manually. Then the measurement starts. As

previously mentioned, the fluids are contained in

deformable balloons. Pistons are used to press the

balloons and put the fluids to movement. In our case,

we have 3 pistons: "sample piston", "buffer piston"

and "probe piston". The pistons motions, and

therefore the fluids flows, are driven thanks to a

computer program. The program also controls the

incubation times and washing duration. When the

biochemical reaction is finished, an ESE

fluorescence measurement unit is used to detect the

possible presence of CMV.

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104

Figure 2: View of the disposable cartridge.

Figure 3: View of the reader/actuator.

It must be noted that the disposable cartridge was

designed using materials and micromanufacturing

processes compatible with a low-cost industrial

production.

3 IMMUNOFLUORESCENCE

DETECTION OF CMV

3.1 Bio-functionalization

Chips are incubated in a solution of 11-mercapto-1-

undecanol (97%) / 16-mercaptohexadecanoic acid

(3%) overnight at room temperature (RT).

Then, 40µl of EDC/NHS (1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride/

N-hydroxysuccinimide) are added on each surface

and incubated during during 30 min at RT. This step

is necessary to activate C11/C16 layer.

The surfaces are then rinsed by 1X PBS

(phosphate buffer saline) and human polyclonal

antibodies (PAbH) are incubated on the chips during

1 hour at room temperature. To ensure optimal

grafting of the PAbH, antibodies are diluted in an

acetate buffer at 0,1mg/ml, pH 5.

Surfaces are then rinsed with 1X PBS and

C11/C16 layer is deactivated using 40µl

Ethanolamine-HCl (1 M pH 8.5) during 30 min à

RT. After a last rinsing by 1X PBS, biochips can be

used.

The fluorescent probe is composed of an anti-

CMV Mouse IgG coupled to an Cy5 - anti Mouse

Goat IgG.

3.2 Experimental Detection of CMV

Immunofluorescence detection of CMV antigen was

experimented in 3 steps.

In a first time, functionalized microscope slides

were used in order to test the biosensor alone. For

this, commercial CMV antigens were used as

biological samples. Six round gold surfaces were

deposited onto the slides and various biochemical

structures were tested as depicted in figure 4. It can

be seen from this figure that when the complete

antibodies-antigen combination is used, the

fluorescent signal is rather high. However, the

different fluids were applied by means of

conventional syringes and the measurements were

not perfectly reproducible.

Figure 4: Biosensor testing with antigen solutions.

DETECTION OF THE CYTOMEGALOVIRUS - A Mobile Device and a Disposable Cartridge for Detection at the

Patient's Bed

105

In a second time, we tested the specificity of the

sensor with various viral proteins obtained from

infected MRC5 cells. This experiment was done on

microscope slides. The result is shown on figure 5

where ADV, CMV and DS stand for adenovirus,

cytomegalovirus and commercial antigen

respectively.

Figure 5: Specificity of the bio-recognotion.

In a third time, disposable cartridges were used

together with the mobile reader. The idea was to test

the complete device with CMV infected cells. This

time, measurements show that the ratio between

positive and negative sample was of the order of 3 as

it can be observed from figure 6.

Figure 6: Complete device testing with CMV infected

cells.

4 DRIVING SOFTWARE AND

FURTHER DEVELOPMENTS

The device presented here is driven by means of a

computer. Fluids flows and optical detection can be

monitored by the computer. Figure 7 shows the

control window of the computer program.

Figure 7: Driving window of the computer program.

In order to enhance the immunofluorescence

reaction, fluids are driven as follows. First, the

biosensor surface is rinsed with the buffer. The

"sample piston" is actuated so that the reaction

chamber is filled. We wait a few minutes so that

viral proteins can be trapped onto the biosensor

surface. Then the "sample piston" is actuated again

in order to fill the reaction chamber with a new

sample volume. After a few minutes of incubation,

the process is iterated until the "sample balloon" is

empty. Rinsing the surface with the buffer is

performed in one go. Then, the "probe piston" is

actuated in the same manner as the "sample piston".

At the end, the biosensor's surface is rinsed with the

buffer and the fluorescence measurement is

performed. The automation of the measurement is

depicted in figure 8.

Figure 8: Control of the fluid flows and incubation times.

A measurement window is used to inform the

operator on the measurement step being processed.

It includes various parameters like the patient's

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name, the name of the virus (we will comment later

on this aspect), the temperature controls and the

virus detection result (see figure 9).

One important aspect concerns the fact that all

the fluid required for the immunofluorescence

detection are contained in the disposable cartridge.

In this way, it is possible to fabricate specific

disposable cartridges for specific virus screening.

We may also envisage the detection of other kind of

biological entities. In fact, we plane to equip the

cartridges with bar codes that will be read by the

reader/actuator. The latter will then tune itself to the

right opto-fluidic parameters such as liquid flows,

incubation times and optical detection threshold. The

computer program offers the possibility to set-up

opto-fluidic parameters for each kind of virus as it

can be seen from figure 10.

Figure 9: Control window.

Figure 10: Setting the opto-fluidic parameters for each

specific virus.

Although this multi-virus arrangement is

particularly interesting in the cases where only one

mobile reader can be used, more specific application

may require a more compact and single virus

detection device. This is the case for CMV screening

for which the device will be used at birth in the care

room next to the delivery room. We are now

working on the ergonomic aspect of this specific

application.

The last issue that must be taken into account is

the time required for the test to be performed. Up to

now, a bit less than 1 hour is required from the

moment when the cartridge is inserted in the reader

and the moment when the result is displayed. It is

much more rapid than a diagnosis in a virology

laboratory (we mean taking into account the gastric

liquid sampling, the packaging of the sample, the

transportation to the virology laboratory and the

diagnosis). However, it can still be improved by

means of acoustic accelerating techniques we are

working on at the moment (Kardous, 2010).

5 CONCLUSIONS

In this conference, we have presented a mobile

device used to screen CMV at the newborn's bed.

Experimental results show a signal to noise ration of

about 3 which is enough for screening purposes. The

fact that all the required fluids are contained in a

stand alone disposable cartridge make the system

easy to transpose to the detection of various

pathology vectors. Our present work deals with the

study of such detections together with the set up of

an ergonomic biological sampling system that fulfill

the requirements of clinical use.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the support

of the French Agence Nationale de la Recherche.

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