Transferrin Electronic Detector for Iron Disease Diagnostics

IEEE SENSORS 2006, EXCO, Daegu, Korea / October 22-25, 2006

Transferrin

Electronic Detector for Iron Disease Diagnostics Brissot P., Loreal 0.

Girard A., Bendriaa F., De Sagazan 0., Har Le Bihan E,Salaun A.C.,Mohammed-Bral

INSERM U522 Universite de Rennes Rennes, France

IETR Universite de Rennes Rennes, France EMAIL: aurelie.girard, france.le-bihan@univ-re Abstract- Field Effect Transistor (FET) structure with suspended bridge shows the ability to detect proteins with very high sensitivity. This sensor is tested for the transferrin, a specific blood plasma protein, relevant in iron disease diagnostic. This system is based on field effect transistor with a suspended bridge used as a gate electrode. The sensitive layer is made of silicon nitride as in the ISFET (Ion Sensitive Field Effect Transistor) technology. The surface micro-technology ensures a small height suspended-bridge (O.5,um) composed of poly-silicon insulated with silicon nitride. To improve sensitivity and to limit biofouling, proteins are selected by antibodies covalently bound to organic layers. The specific charge of the fixed transferrins translates the transistor transfer characteristics. For low concentration, resulting shift is important and confers a good sensitivity to our electronic transducer. I. INTRODUCTION

The ability to determine the quantity of biomolecules is important in medical, pharmaceutical and biotechnological research. Due to semiconductor technologies, a great progress in diminution of costs and miniaturisation is achieved. Many types of biosensors exist and are able to detect biomolecular interaction. Sensitive identification and measurement of levels of circulating proteins or proteins in blood is clearly a priority and means detecting proteins in very small quantities. This technic currently involves the joint problems of the selection and the detection. Protein chips, those based on antibody arrays, may provide inexpensive, rapid screening methods for these

applications.

(acoustic wave or micro-balance, ...) types. The most popular transducers are systems with electrodes like ion sensitive field effect transistors (ISFETs), solid state electrodes, Clark electrodes and chemically sensitive resistors [4] [5]. Here we propose an electrical detection device, with a very particular electrode, developing as a biosensor based on well-known ISFET principle. Moreover, our analytical device combines a biologically sensitive element with a chemical transducer to selectively detect the presence of specific compounds, here transferrins. II. TRANSFERRINS Iron is an essential nutrient for human body. Too less or too much iron can compromise many important functions and can cause critical damage to organs [6]. Transferrins (Tf) are iron-binding glycoproteins. They are present in various body fluids. Transferrin is the only carrier of iron. It circulates in blood plasma and is produced by the liver. It contains 679 amino acid residues and has a molecular weight of 79kDa. The transferrin protein measures lOnm and is negatively charged at blood pH (pH = 7.4). It has two metal-binding sites and allows to transport iron from blood to organs in need [7]. The binding and release of iron by Tf involves several factors, for example, pH and temperature. The level of Tf in blood is important to detect iron diseases in the iron metabolism: overload or anemia. Plasma concentration of Tf is stable from 2g/L to 3g/L. Its measurement is difficult to realize and expensive. It consists on a calculation of the transferrin saturation coefficient in iron. The objective of the present work is to show the ability of our device to detect such heavy and low concentrated protein with enough sensitivity and rapidity. Some processes to screen for these types of disease exist like radioactive (RIA), luminescent (LIA), fluorescent (FIA) or enzymatic (EIA) tests as ELISA (Enzyme-Linked ImmunoSorbent Assay). Our sensor is based on the same binding principle but at the difference there is no need to have labels on proteins.

This is the reason why immonusensors are becoming increasingly important [1] [2] [3]: they use speedier methods of detection and selection of small protein quantities. Already, many traditional approaches use antibodies and are based on detectors which operate through highly specific biological recognition sites. However molecular detections are commonly helped by labelled target molecules like fluorescence or radioactive tags for example. These methods cause problems because they are expensive and difficult to implement. To overcome these drawbacks, a label free detection mode is III. DESCRIPTION AND DETECTION PRINCIPLE necessary. Transducers for label-free immunosensors manufactured A. Description The SGFET (Suspended Gate Field Effect Transistor) is with semiconductor technologies may be of electrochemical based on the ISFET principle and technology. or mass (potentiometric, amperometric, conductometric, ...)

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position in the semiconductor versus the mid-gap, Qsc is the space-charge in the semiconductor, C is the total capacitance between the gate material and the semiconductor, e0, is the thickness of the insulator that is both the air-gap and Si3N4 here, (x) is the charge in the insulator at a distance of x from the gate. Any variation of the ambience in the air-gap leads to a variation of the total charge in the insulator and a possible variation of its distribution. Moreover some chemical reactions at the inner surface of the gate material and at the surface of the channel or the passivating film deposited on it, can occur leading to a variation of ¢3MS. Fig. 1. Up-view of SEM observation for the SGFET structure.

IV. FABRICATION

The use of ISFET to measure pH and to sense multiple ions is well known [8] [9]. The main part of an ISFET is the ordinary Metal Oxide Silicon Field Effect Transistor (MOSFET) with the gate electrode replaced by a chemically sensitive membrane, solution and a reference electrode (RE). In this device, the gate insulator is directly in contact with the solution. Inspired by this architecture, SGFET, presented in figure 1, is an ISFET with a suspended gate, included in the structure, as reference electrode. This permits to sense charge into a very confined space to improve the sensitivity. Moreover, the architecture is portable, and its use in pH tests [10] shows a really higher sensitivity than the ISFET one. B. Principle Like in MOSFET, the channel resistance in SGFET depends on the electric field perpendicular to the direction of the current. Charges from solution accumulate on top of the insulating membrane and do not pass through it. The dependence of the interfacial potential on the charge concentration can be explained with the site-binding theory presented by Yates [11]. To detect the charges on the sensing membrane, a voltage is applied on the gate to create the electrical field between the bridge and the channel. Then the drain current versus the gate to source voltage (ID vs. VGS) characteristics of the field effect transistors is measured. During measurements, the drain source voltage (VDS) is kept constant at 2V and VGS is swept from positive to negative voltage. To sum up, any voltage applied to the bridge (suspended gate) is capacitively coupled via the electrolyte to the insulator surface. In the electrolyte, charges on the interface solution/sensors modulate the channel current and causes shifts in the transfer characteristic, thereby modulating its threshold voltage Vth. The threshold voltage Vth of SGTFT can be expressed by the equation 1 and 2.

Vth

= =

Vthdevi + Vcharge Oms + 2)F + Qsc c

JI

Cco

(1) eOx

xp(x)dx (2)

Where q5MS is the difference between the work function of the gate material and the semiconductor, OF is the Fermi level

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Draih

Source ,.........w

.. .... ..........

...

Gate

Silicon Silicon P-doped

poly-Silicon

........... ...........

Silicon Nitride Aluminium

I

I

Photoresist

Silicon Oxyde

Fig. 2. Schematic configuration of the SGFET.

The device, see figure 2, is fabricated on < 100 > oriented silicon wafer with low N-type doping. First steps are similar to classic MOSFET technology. The difference comes from the necessity to create a "bridge", the gate, in poly-silicon. We use a silicon nitride, Si3N4, layer to use it as a sensitive layer. To suspend the gate, a sacrificial germanium layer is deposited by LPCVD between the Si3N4 layers. Two 45nm of Si3N4 layers are deposited by LPCVD (Low Pressure Chemical Vapor Deposition) under and on the gate for isolation. The germanium layer is 500nm thick and it is removed at the end of the process. Due to its great etch selectivity against silicon oxide, silicon nitride and poly-silicon and to the unstable germanium oxide, allows us to remove very qucikly and efficiencely the germanium. This etch has a rate of 1, 2,um/min in H202 held at low temperature 900C. Several transistors are fabricated with different gate sizes. There are three channel dimensions: 15,um by 25,um, 30,um by 80,um and 15,um by 130,um. The mechanical resist of all the "bridges" was studied for immersions, manipulations, stress. To provide electrical and chemical isolation, the inactive device area is passivated with a SiO2 (lOOnm) and a photo-

lithographic resist layer.

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IEEE SENSORS 2006, EXCO, Daegu, Korea / October 22-25, 2006

V. FUNCTIONALIZATION BY SURFACE MODIFICATION AND BIOMOLECULES IMMOBILIZATION The surface chemical modification can be realized from the NH2 groups on surface which is a starting silicon nitride surface after the etching of germanium. The NH2-terminated surface can attract negative charges. This effect is valid in air or in electrolyte solution where negative charged particles are effectively physio-adsorbed [12]. Negatively charged biomolecules such as transferrins might be also directly detected if they are present by adsorption on the sensitive layer. But for our part, a covalent binding to the surface is chosen. The selection mode is based on the transferrin antigenic power. The antibodies immobilization on the sensitive surface separates transferrins from other plasma molecules.

A. Materials Anti-human tranferrin, human transferrin and glutaraldehyde are provided by Sigma-Aldrich,inc; APTS (3-Amino Propyl Trimethoxy Silane) by Fluka and Riedel-de Haen Company. B. Methods Biological receptors, i.e. enzymes, antibodies, cells or tissues, can be immobilized in a thin layer at the transducer surface using different procedures. The most common immobilizations are done by entrapment or adsorption like in ELISA tests. we have chosen covalent binding because it permits to obtain more stable and more reproductible measurements even if this type of attachment is more complicated to realise. NHS-4-...

H,

H~

..........

SiN

H C H

C

C

H.

H

H

C ~~, C H.

-1 ,Oxlo'

-

--

-8,oxlo-, -

Unfunctionnalized

Glutaraldehyde Glutaraldehyde + Antibodies

-6,OX10 -

-4,OX10 -' _2.OX 1O'5 0,0

-

0

1

2

3

-4

-5

-6

-7

8

9

10

---I

-11

Vgs (V)

Characteristics in air of the transistor before, and after the Fig. 4. glutaraldehyde binding and the anti-transferrins binding.

For the second part of the test, transferrins are linked to the antibodies by immersion in PBS. The presence of the grafted transferrin is confirmed by the shift of the SGFET transfer characteristic. We obtain a shift for a detection in air, of 202mV for a transferrin concentration of 1,ug/mL, representing the transferrin blood concentration divided by 20. The measurement was done for a concentration of 1O,ug/mL for the antibodies. This first test in air was necessary to prove the ability to detect transferrin. The transfer in the air is illustrated by the figure 5.

H

H

VI. RESULTS Each step of the functionalization and of the tests is followed by a measurement, like explained in the part V. As each modification is responsible of a new charge under the gate, a new reference curve is mandatory for further comparisons and experiments. The first test presented here was done to verify if the transfer characteristic of our sensor shifts for each functionalization step. If it is the case, it will prove that glutaraldehyde and antibodies are linked on the sensitive area. For this test, only measurement in air was noted down because the only needed information expected is functionalized or not. The figure 4 shows the characteristics of each step of the silicon nitride functionalization. The shifts between them prove the binding of glutaraldehyde first and then of antibodies, and valid this first step.

AO'FE

-1 ,6x104-

Fig. 3. Functionalization of the surface with glutaraldehyde and antibobies.

First of all, a glutaraldehyde layer is evaporated on the device [13] and bound on the NH2-terminated from the nitride silicon by its carboxyl group, see figure 3. The operation is done at the room temperature during half-hour. After, the device is rinsed several times and a transfer characteristic is noted down. The antibodies solution is prepared from SigmaAldrich powder and PBS. Phosphate Buffered Saline is a solution with a constant pH at 7.4 as blood and a constant ionic force. The solution is pipetted onto the chip and incubated for 30 minutes at the room temperature. Then the sample is rinsed several times in PBS and the transfer characteristic which becomes the new reference is measured.

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s

-1,4x10

-

-1,2x1

-

-1,OxI1

-

-8.xO1 o-6,oxl o0

-

-4,Oxl O'

-

-2,Oxl O

-

--------

Glutaraldehyde + Antibodies Glutaraldehyde + Antibodies+ Transferrin

0,0 -

2.,0x1

-4

5

-6

7

-8

Vgs (V)

Fig. 5. Visualization of the translation of the transistor transfer characteristics before and after the binding of transferrins (measurement in air).

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Others tests were completed but this time with a controlled

IEEE SENSORS 2006, EXCO, Daegu, Korea / October 22-25, 2006

environnement because the device is very sensitive to all ions. This is why all the detections are done at the same pH, first to be sure that the transferrin will not be denatured and remains in the same conditions. It is also necessary to maintain the protein function and to stabilise its charge in a buffer solution to be sure to detect the charge of the molecule. For this, we use the same functionalization, protocol and concentrations. The only difference with the other tests presented comes from that all the measurements are done in PBS at pH = 7.4. In the figure 6, the shifts between glutaraldehyde and antibodies represents 292mV at -Id = lO,uA for an antibodies concentration of l,ug/mL, and the other between the antibodies and the transferrin represents 27OmV at Ids = lO,uA for a transferrin concentration of l,ug/mL. Moreover, the positive shift of Ptype transistors means the presence of negative charge that is transferrin charge in these conditions. These tests prove the ability to detect a very low transferrin concentration with high sensitivity in a physiologic solution. -2,OX100-

-1,5x10-

ns

-

[3] M. Bengtsson T. Laurell J. Emneus J. Yakovleva, R. Davidsson. Microfluidic enzyme immonusensors with immobilised protein a and g using chemiluminescence detection. Biosensors and Bioelectronics, 19 :21-34, 2003. [4] Piet Bergveld Geert Besselink. Affinity biosensors. Humana Press, 7 :173-186, 1998. [5] M. Saleemuddin R. Ulber T. Scheper M. Farooqi, P. Sosnitza. Immunoaffinity layering of enzymes. Applied Microbiology and Biotechnology, 52 :373-379, 2004. [6] 0. Loreal P. Brissot, M.B. Troadec. The clinical relevance of new insights in iron transport and metabolism. Current hematology reports, 3 :107-115, 2004. [7] B. Ingelman C.B. Laurell. The iron-binding protein of swine serum. Acta Chemica Scandinavica, 1 :770-776, 1947. [8] P. Bergveld. Thirty years of isfetology. Sensors and Actuators B, 88: 120, 2003. [9] I. Gracia C. Cane and A. Merlos. Microtechnologies for ph isfet chemical sensors. Microelectronics Journal, 28 :389-405, 1997. [10] A. C. Salaun T. Mohammed-Brahim F. Bendriaa, F. Le Bihan and 0. Bonnaud. Highly sensitive suspended-gate ion sensitive transistor for the detection of ph. Proceedings of SPIE, 5836 :433-440, 2005. [11] Samuel Levine David E. Yates and Thomas W. Healy. Site-binding model of the electrical double layer at the oxide/water interface. J. Chem. Soc., 70 :1807- 1818, 1974. [12] A. Elaissari J.P. Cazenave J.C. Voegel-P. Schaaf V. Ball, P. Huetz. Kinectics of exchange processes in the adsorption of proteins on solid surfaces. Proc. Natl. Acad. Sci., 91 :7330-7334, 1994. [13] N. Jaffrezic. Internal communication ietr. 2005.

-1,Ox1 0-F-

-5,Ox1 0-8 -

/ *..| - G~~~~lutaraIdehyde

0,0

---- Glutaraldehyde ---Glutaraldehyde

_ _------

-

-

Antibodies Antibodies + Transferrin

|

4

-6

5

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

-8

Vgs (V)

Fig. 6. Visualization of the translation of the transistor transfer characteristics after each steps of the detection protocol (measurement in PBS).

VII. CONCLUSION We see that our sensor has a good isolation and is able to catch and detect transferrins under its gate. This is permitted by the surface functionalization of the silicon nitride. The small size and capability of the microbridge for sensitive, label-free, real-time detection of an adapted range of biochemical species can be useful in analysis and in diagnostics. We can conclude that our system resists to several immersions and drying in a biological compatible solution, promises to be selective and gives an instant result in opposition to the others tests especially the labelled ones. SGFET are promising candidates for single use sensors. ACKNOWLEDGMENT

The authors would like to thank the entire IETR laboratory as well as the INSERM unit 522 for their helps and advices. REFERENCES [1] G.K. Budnikov E.P. Medyantseva, E.V. Khaldeeva. Immunosensors in biology and medecine analytical capabilities, problems and prospects.

Journal ofAnalytical Chemistry, 56(10) :1015-1031, 2001.

[2] K. Kramer B. Hock, M. Seifert. Engineering receptors and antibodies

for biosensors. Biosensors and Bioelectronics, 17 :239-249, 2002.

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