CHEMICAL AND/OR BIOMOLECULAR ANALYSIS

INTEGRATED SYSTEM AND METHOD FOR

MANUFACTURING THE SAME

The present invention relates to a chemical and/or biomolecular analysis integrated system. The invention further relates to a method for manufacturing the same system.

More particularly, the system suggested according to the present invention permits realising a complete chemical and/or biomolecular analysis system on a single support, which is simple and reliable, and that can be obtained by a manufacturing method that can be carried out at low temperature, thus being reliable and not expensive.

The suggested invention is included in the specific field of Lab on Chip systems, able carrying out complex analyses in an automatic and quick way, using small amounts of the sample analysed, that can be used for DNA analyses for genetic recognition, for in situ toxicological analysis of substances destined to alimentation, for which strict rules exist for quality control.

Substantially, the invention suggests a technology for chemical and/or biomolecular analysis that can be applied in different biotechnology fields.

The solution suggested according to the present invention permits integrating, on a single support, basic operation of a chemical and/or biomolecular analysis, such as separation of substances to be analysed, movement of substances toward reaction and/or analysis sites, control of reaction mechanism to be eventually carried out on the device for preparation or marking purposes, verification of reactive events course exploiting all the spectroscopic and chromatographic features of analytes, detection for analytical purposes of fluorescence, chemiluminescence and bioluminescence phenomenons that can be referred to analyte (not chemiluminogenogenic compounds can be measured after a suitable marking).

Manufacturing of the whole system is based on the use of thin film and thick film technologies, usually used in microelectronic field that, as it is well known, permit realisation of micrometric geometry structures on different layers (different material and shape) and having dimensions of different cm ² ; particularly, it will be possible using them as laboratory

slides support on which different materials will be deposited, for deposition of sample and its separation/migration toward active elements.

Device suggested according to the present invention can be used for structural characterisation of materials deposited by measurement of diffusion though the same of fluorescent molecules having known chemical-physical features.

With respect to the known techniques, requiring for each procedure a dedicated apparatus, possibility obtained according to the invention of having at disposal a compact instrument, with all the main analysis procedures, permits obtaining different advantages, among which: easiness of use, speed of analysis, portability, and possibility of remote control of procedure for analysis in hostile environments.

It is therefore specific object of the present invention a chemical and/or biomolecular analysis integrated system comprising a support, on a face of which a photo sensor is realised, and on the same face, or on the opposite face with respect to said photo sensor, it is positioned sample to be analysed, said photo sensor providing a current proportional to the luminous power emitted by chemiluminescent, bioluminescent or fluorescent samples, reading of photocurrent permitting a quantitative measurement of analysed sample, in case it is a fluorescent sample, or measures carried out by absorption, the system being illuminated by a suitable electromagnetic radiation, being it possible determining substances having intrinsic emission features, or being it possible actuating marking procedures, making it more specific the determination. Particularly, according to the invention, said support can be comprised of a laboratory slide.

Preferably, according to the invention, said photo sensor is an amorphous silicon photo sensor, particularly comprised of overlapped layers so as to realise a p-doped/intrinsic/n-doped structure, i.e. a structure with two electric contacts, that, if suitably polarised, make it active diode as photo sensor.

Always according to the invention, said system comprises means for separation of substances to be analysed, means fro moving substances toward reaction and/or analysis sites, and means for controlling mechanism of reaction occurring on device for preparation or marking purposes, means for verification of course of reaction events exploiting spectroscopic and chromatographic features of analytes, means

for detecting, for analytic purposes, fluorescence, chemiluminescence and bioluminescence, or absorption phenomenons that can be referred to analyte.

Particularly, according to the invention, preparation of sample for analysis can be directly carried out on the device by thin layer chromatography and/or by an electrophoretic or dielectrophoretic travel methods and/or by chemical procedures, including the biomolecular amplification with chain polymerase (PCR) induced by thermal cycles.

Still according to the invention, molecular movement and possible separation of analytes present within the mixture can be carried out according to thin layer chromatography methods using the suitable movable phases, and/or by help of electric fields having suitable intensity and frequency, applied on conductive electrode structures, deposited on the substrate surface by thin film techniques (evaporation, sputtering, ecc.) and suitably conformed by photolithographic techniques.

Furthermore, according to the invention, possible thermal cycles can be carried out in substrate zones on suitable heaters, comprised of thin film resistors manufactured by microelectronic planar techniques, with temperature sensors comprised of metals and/or amorphous semiconductors couplings on thin film.

Still according to the invention, verification of course of reactive events can be carried out, by thin film sensors realised on the same substrate that can monitor variation of a chemical-physical parameter, such: absorption or emission, electric conductivity, ecc. According to the invention, above-mentioned operation can be managed automatically by a single electronic control system connected with the support.

The invention further relates to a method for manufacturing the above system, providing all or some of the following steps: - deep vacuum Physical Vapour Deposition (PVD) of a metallic thin film (< 1 micron) (e.g. titanium/tungsten alloy) and conformation, by photolithography and selective chemical attack, of heating resistor and/or of lower contact of diode for measuring temperature, and/or of lower contacts of photodiodes for monitoring electrophoretic travel, and/or column tracks of photodetector matrix;

- deposition of diode in hydrogenated amorphous silicon (a. Si: H) by Chemical Vapour deposition (CVD), with multiplayer structure

n-doped(intrinsic/p-doped, with a total thickness < 1 micron, and conformation, by photolithography and chemical/physical attack in reactive plasma (RIE) of areas of devices on geometries of corresponding metallic contacts, - deposition of an insulating layer (e.g. silicon nitride, silicon oxide) and realisation, by photolithography and selective attack, of electric interconnection holes (via-hole) on a-Si active devices,

- deposition a conductive and transparent thin film (TCO) by deep vacuum sputtering, and conformation, by photolithography and chemical attack, of geometry of upper electric contacts of photodetectors in the system central zone, and of matrix line tracks,

- passivation of the system by CVD deposition of a silicon nitride layer;

- realisation of parallel metallic electrodes by thin film techniques or conductive paste serigraphy, by which it will be possible inducing suitable electric fields for movement of substance to be analysed by electrophoresis;

- deposition of material on the support face provided with photosensors, or on the opposed face, apt confining sample mixture or acting as fixed phase for a chromatographic or electrophoretic travel. Choice of material will be made on the basis of analytes to be determined. The present invention will be now described, for illustrative but not limitative purposes, according to its preferred embodiments, with particular reference to the figures of the enclosed drawings, wherein: figure 1 shows a basic scheme of a system according to the invention; figures 2, 3 and 4 show graphs relevant to experiments carried out employing the system of figure 1 ; figures 5a and 5b are views of a prototype of a system according to the invention; figures 6 and 7 show graphs relevant to photocurrent of one of amorphous silicon photosensors of matrix, during chromatographic travel of substances contained within a detergent and an marker ink:; fixed phase : silica gel : movable phase : ethylic alcohol; and figure 8 shows a second embodiment of the system according to the invention.

Observing first figure 1 , it is shown a scheme of operation of the system according to the invention, providing an amorphous silicon photo sensor Iprovised on a laboratory slide 2, on the opposite face of which, aligned with sensor 1 , is positioned substance 3 to be analysed. In the specific case, substances to be analysed are fluorescent substances; illuminating substrate with ultraviolet radiation, excited fluorescent pigment will emit visible light.

If not fluorescent substances are analysed, analysis can occur marking before analyte with standard fluorchromes. Light emitted by fluorchrome, excited by ultraviolet radiation, produces a current on sensor 1 , which is proportional to the incident luminous power. Reading photocurrent of sensor 1 , it is thus possible receiving a quantitative response of the analysed substance.

Photodetectors employed for sensor 1 are comprised of amorphous silicon layers, stacked so as to realise a p-doped/intrinsic/n- doped (p-l-n) structure.

Thus, it is a device with two electric contacts that, if suitably polarised, make diode active as photo sensor. A suitable designing of structure, and particularly of thickness and of layer optical absorption, permits maximising response of photo sensor 1 at the fluorchrome emission wavelength.

Thus, different advantages are obtained, such as alignment between substance 3 to be detected and photodetector, and filtering of UV excitation radiation with respect to the fluorchrome emission light, thus permitting elimination of optical systems assemblies and the consequent reduction of costs.

To investigate efficiency of this system and characterising it sensitivity, it was carried out a series of experiments for p-l-n structures, using growing concentrations of marked DNA in a solution and measuring values of sensor photocurrent. It was chosen "Alexa Fluor 350" fluorchrome, having an excitation peak at 350 nm and an emission peak within visible at 450 nm.

Course of photocurrent in function of concentration shows an excellent linearity and a minimum detectable DNA concentration of e nmol/l. taking into consideration specific activity of fluorchrome in inventive marking process, this value corresponds to a minimum surface fluorchrome density of about 50 fmol/cm ² . This value can be compared

with the result obtained with standard, but much more expensive, laboratory apparatuses.

Basic idea has also been applied in the "Food Quality Control" field for detection of micotoxines. Particularly, photocurrents induced in sensor by solutions with different concentrations of Standard Ocratoxin A

(OTA) was measured. This dangerous micotoxin can be for example found in wine, wherein the limit set by the law is of about 0,1 ng.

Still for illustrative, but not limitative purposes, a prototype of a system integrated on glass is shown in figures 5a and 5b, for particular application for chromatographic analysis. A silica gel layer (fixed phase) is deposited on a face of a glass substrate, while a row of 16 hydrogenated amorphous silicon (a-Si:H) photosensors is realised on the opposite face (figure 5a). lower contact of devices is realised by a single conductive and transparent oxide film (TCO), while upper contacts are realised as a metallic thin film. System is electrically connected with outer electronic circuit by a suitable metallic track geometry up to the glass edge, and a "slide" standard connector (figure 5b).

A small amount of the sample to be analysed is placed on silica gel, close to the edge, from which a mixture of suitably chosen solvents (movable phase) is made flowing by capillarity, determining migration of different components of mixture according to modes determined by molecular features (dimensions and charge).

Sample components will be each one deposited at a different distance from the starting position, thus individuating a band structure, which is a feature of the analysed sample (figure 5a).

As already mentioned in the above, in case substances to be analysed are naturally fluorescent, illuminating substrate by ultraviolet radiation, excited fluorescent pigment will emit visible light. Reading photocurrent of sensors on the opposed surface of slide it is possible determining position of bands of chromatographic travel, comparing the result with reference samples, it is possible confirming analyte features. In case not fluorescent substances are analysed, recognition can occur marking before analyte by standard fluorchromes or, as alternative, fluorescent gels can be used for the fixed phase, and in this case, system will provide a negative image of the analyte band structure.

Obviously, if analyte shows chemiluminescence of bioluminescence (typical of analyte or induced by marking the same, the use of an outer source will be not necessary.

A real time measure of substance migration through fixed phase can be carried out by the same system, monitoring photocurrent of different sensors. Knowing distance between devices it is possible knowing analyte displacement speed that is a feature of the same analyte and/or of the fixed phase employed.

A process comprising the following steps is carried out for realisation of prototype of figure 5 (application to thin layer chromatography):

- sputtering deposition of an Indium and Tin Oxide layer, thick 1 micron, acting as TCO;

- deposition of hydrogenated amorphous silicon (a-Si:H) by Chemical Vapour Deposition (CVD) technique, with p-doped(intrinsic/n- doped multiplayer structure, with a total thickness of 1 micron, and thermal evaporation under vacuum of a metallic thin film. Conformation by photolithography and chemical/physical attack in reactive plasma (RIE) of device areas; - spin coating deposition of and insulating layer and realisation of electrical interconnection holes (via-holes), by photolithography and selective attack, of a-Si active devices;

- deep vacuum sputtering deposition of a titanium thin film and conformation, by photolithography and chemical attack, of metallic track geometry from upper electrical contact of photodetectors to the glass substrate edge for connection with outer circuit managing the process, employing standard "slide" contacts;

- positioning of fixed phase of chromatography travel (silica gel) on glass surface opposite with respect to sensors. Prototype shown in figures 5a - 5b was subjected to two chromatographic analysis tests.

In a first test, it was determined presence of fluorescent substances in a tissue detergent sample, while in the second test it has been detected presence of fluorescent pigments in a marker pencil. Graphs of photocurrent of one of amorphous silicon photosensors of matrix of substances contained within detergent and marker are shown in figures 6 and 7, during the chromatographic travel,

using ethylic alcohol as movable phase. Carrying out tests, silica gel plate was illuminated by ultraviolet light. Current drop evident from figure 6 corresponds to passage above sensor area of fluorescent current contained within soap. Three current increments are evident from figure 7, corresponding to three different components contained within marker colour.

Always for illustrative and not limitative purposes, figure 8 shows an example of integrated system on glass, more complete with respect to the previous one, on which all basic operations of a chemical and/or biomolecular analysis can be carried out, and particularly functions necessary to recognition of DNA fragments.

Substrate 2 can be comprised of a laboratory slide, with dimensions of 2.5 x 7.5 x 0.1 cm ³ , on which structures for the different operations necessary for carrying out the complete analysis are realised. A passivation layer is laid down on said structure, insulating the analysed substance from the active part of the system. Before the molecular recognition process, sixteen different known single helix DNA sequences are immobilised by standard "spotting" techniques on 4 sites corresponding to 16 pixels of a matrix of amorphous silicon photodetectors, directly realised on substrate.

Some fragments of unknown single helix DNA into a solution are inputted on a 1x1 cm ² area, dedicated to molecular amplification by PCR technique, a thin film metallic resistor 5 (e.g. chrome) being realised on said area, with an amorphous silicon diode temperature sensor 6. Resistor and diode electric contacts arrives on an edge of glass for connection with supply outer circuit 7.

After the amplification phase, molecule electrophoretic travel starts, applying an electric field between at least two metallic electrodes 8 placed above the passivation layer. It is possible real time monitoring, during the travel, along a 1 x 3 cm ² area, of molecule passage, by reading photocurrent signal provided by amorphous silicon photodetectors 9 having a 1 x 10 mm ² area.

Once passed this zone, electrophoretic travel of DNA molecules prosecutes through the recognition zone. In case unknown single helix molecule meets, along its travel, complementary molecule among the 16 immobilised sequences, reaction reconstructing double helix (hybridisation) occurs. In this case, molecule ends its electrophoretic

travel, fixing on the corresponding site. By illuminating with a suitable monochromatic light, matrix area of 4 x 4 photodetectors, before and after the possible hybridisation, it is possible recognising the hybridised site and thus type of unknown DNA. The present invention has been described for illustrative but not limitative purposes, according to its preferred embodiments, but it is to be understood that modifications and/or changes can be introduced by those skilled in the art without departing from the relevant scope as defined in the enclosed claims.