Patent Application: US-44029707-A

Abstract:
the present invention relates to a system and process for detection and / or qualitative and quantitative identification of the biological material , such as specific sequences of nucleic acids or proteins as antibodies , present in biological samples . the system is composed by one or more light sources combined with one or more integrated optical photo sensors , or not , and various electronic components , necessary for obtaining / processing of the signal emitted by the metal nanoprobes functionalized with a solution of biological composite , as well as also a micro - controller and a microprocessor , fixed or portable . this photosensor structure is able to detect and to quantify the color variations produced by metal nanoprobes , being this preferentially gold , functionalized by oligonucleotides complementary to specific dna / rna sequences , proteins , as for instance antibodies and / or antigens related with certain disease , or other sample or solution of biological composite , that are to be investigated . the detection and quantification process is based on the response of a photosensor , singular or integrated , based on thin film technology of amorphous , nanocrystalline or microcrystalline silicon and their alloys , as well as the new active ceramic semiconductors , amorphous and not amorphous .

Description:
as previously referred the main components of the system are a monochromatic light source , the described photo sensor and suitable electronics for processing and acquisition of the results . subsequently , a detailed description of one possible configuration of the present invention is made , now that various configurations can exist depending on several factors , such as the type of light source ( laser or light emitting diodes ), type of electronics used for the signal processing , type of photo sensor used ( single or integrated ), etc . a single amorphous silicon photo sensor ( 6 and 7 a ) is used with a pi ′ ii ′ n structure , deposited on a glass substrate , in which the maximum spectral response can be adjusted in a broad range of the visible spectrum such as 530 nm . the light source ( 1 ), is a monochromatic laser of wavelength close to the one above selected , such as 532 nm ( green light ) and a power such as 5 mw , placed perpendicular to the photosensor ( 6 and 7 a ), described previously with a structure such as pi ′ ii ′ n structure , so that the incident light points exactly on the geometrical centre of the photosensor ( 6 and 7 a ), reducing so the amount of reflected light when it passes through the biological liquid ( 5 ). the distance is variable , as a function of the light source power ( 1 ), between 5 cm and 30 cm , preferably being 15 cm . the wavelength of the light source ( 1 ), as for instance 532 nm , corresponds to the optimized spectral response peak of the photosensor , and it is selected in a complementary manner according to the metal nanoprobes used , as previously referred to . light guiding systems can be used , such as optical fibbers , or lenses , in order to optimize the quantity of the light incident on the sample . the light source ( 1 ), as referred previously is pulsed at a frequency as for instance 130 hz , in order to eliminate the noise associated with the ambient light , therefore being able to use continuous light sources ( 1 ) and to make use of electrical or mechanical pulsing ( 2 ) techniques for this incident monochromatic light beam , by using external apparatus ( for example a chopper , in order to pulse the signal mechanically ). also , in order to reduce ambient light effects , optical filters ( 20 ) are to be used which are specifically tuned for this light source ( 1 ), ( e . g ., monochromatic laser of wavelength 532 nm ) to be used in the essay . the photosensor ( 6 and 7 ) is deposited on the bottom part of the structure ( 3 ) and it is optimized for the light source ( 1 ) selected to be used on the test being realized . if shifting the peak of the spectral response of the photosensor ( 6 and 7 ) to another range is needed , then voltage sources ( 19 ) can be used in order to proceed to its polarization and in this way , to shift the spectral response of the photosensor ( 6 and 7 ) to the desired range . this voltage source ( 19 ) is manually regulated , since the polarization of the sensor ( 6 , 7 ) depends on the analyses to be performed . the biological liquid ( 5 ) is placed on the opposite side of the substrate where the photosensor structure was deposited ( pi ′ ii ′ n structure described previously ), gaining the advantage of the liquid not being in direct contact with the sensor , but rather being in contact with the rear side of the substrate that contains the sensor . this fact permits reusing the photosensor , implying a lower cost on the use of the system . on the other hand , the area of contact is large enough to allow rigorous qualitative and quantitative results and so there is no need to use a large quantity of sample , allowing so a cost reduction associated to the sample . the light source ( 1 ), which is located perpendicular to the sensor , will emit a radiation that passes through the biological liquid ( 5 ) placed on the surface backside of the substrate that contains the photosensor . the non absorbed light by the drop of biological liquid ( 5 ) passes through the vitreous substrate ( 6 ) and it is absorbed by the photosensor , which converts the light signal to a photo - current and / or a photo - voltage ( electrical signal ). the electric signal acquired by the photosensor ( 6 and 7 ) is compared , subtracted , amplified or filtered by electronic circuits external to the sensor , using synchronous amplifiers ( 4 ), regulated to the same frequency as the light source ( 1 ), thereby obtaining the results from the electrical signal generated by the photosensor ( 6 and 7 ). the detection response ( r det ) is measured as a function of the variation of current or voltage of the device , specific for each wavelength and light intensity used , which it is the difference between the reference values , meaning , the value of the reading of the light beam projected directly onto the sensor ( r ref ) and the value of the reading after having placed the biological liquid onto the sensor ( r adn ). various types of biological sample can be used on the system , and therefore the case of nucleic acid ( dna / rna ) identification is pointed out as study case . the dna / rna sample is extracted from blood , saliva , etc ., by commonly used processes , for example existing purification kits . subsequently , the sample is purified , and an amplification step might or might not be necessary . in the case of rna the amplification step is not necessary . the purified dna / rna is therefore mixed with nanoparticle probes , where the nanoparticles are made of gold or other metals , in accordance with the following process : the dna / rna , combined with the gold nanoparticle probe , is exposed for 10 minutes to 95 ° c . in order to denature de double chain and / or secondary structures of the dna / rna . immediately after , the sample is left to cool down for 30 minutes at room temperature allowing the denatured dna / rna to hybridize specifically with the gold nanoparticle probe . an electrolyte ( i . e . nacl ) is then added up to a final concentration of 2m . after 15 minutes at room temperature ( between 5 and 45 ° c .) it is possible to observe the results as a function of the calorimetric change . then , the biological liquid , containing the nucleic acid samples to be detected or identified together with the respective gold nanoparticle probe , is placed on the photosensor backside of the substrate , and the result revealed . therefore , when various solutions containing gold nanoparticle probes and dna / rna test are being prepared , and after the addition of a salt it is possible to observe the results , as a colour change in the case of being negative or with the colour remaining the same in the case of being positive , which it is characterized by the photosensor having different electrical signals for each case . this corresponds to two different linear behaviours of the photo - sensors , respectively for the colour change ( associated to the wavelength ) and light intensity change of the light ‘ emitted ’ by the nanoprobes . according to what was previously described in the detailed description , the configuration shown in fig1 was chosen for this example . a single amorphous silicon photosensor was used , deposited on a vitreous substrate ( 6 ); the light source ( 1 ) chosen was a solid state laser with a wavelength of 530 nm , which is associated to the maximum spectral response of the photosensor [ 29 ]. the light source ( 1 ) is mechanically pulsed at a frequency of 130 hz and the photo voltage generated by the photosensor is measured with the help of a synchronous amplifier ( 4 ), using the pulsing frequency of the laser as a reference . the non - absorbed light ‘ emitted ’ by the nanoprobes passes through the vitreous substrate ( 6 ) and it is absorbed by the photosensor , which converts the signal to a photo - current or photo - voltage . since the photosensor works in the photovoltaic mode , no power source is necessary to feed it . the light emitted by the laser works simultaneously to induce variation on the biological liquid absorption , as well as to activate the photo sensor . the detection response ( r dft ) was measured as a function of the variation of current of the device , being the difference between the reference values , meaning , the difference value between the reading of the light beam projected directly onto the sensor ( r ref ) and the value of the reading after having placed the biological liquid onto the sensor ( r adn ). although the values of the parameters r adn , r ref had been read in the photo - current mode , the obtained results have units of voltage , due to the conversion performed by the synchronous amplifier ( 4 ). the results have a strong linear behaviour as a function of the probe concentration , turning possible to make a proper approach with a correlation factor , for instance better than 0 . 97 . this fact satisfies lambert - beer &# 39 ; s rule , where the increase in probe concentration it translates into an increase of incident light absorption , a fact that maximises the difference of values between r adn e r ref . the proposed system was effectively applied to dna detection . the probe was designed to be complementary to a specific genomic region of the rna polymerase β - subunit of mycobacterium tuberculosis ( tuberculosis agent ). the results were attained by measuring the responses of the sensor to the colour changes of a blank solution , non - complementary dna , and complementary dna . in this test , four solutions were prepared , keeping the au - nanoprobe concentration constant at 2 . 5 nm , namely : buffer solution ( solution1 ), which it is used to prevent major variations on the solution &# 39 ; s ph ; sample with non - complementary dna ( solution2 ); samples with complementary dna from m . tuberculosis ( solutions 3 and 4 ). results show that all liquids used absorb the incident radiation . this happens since the photosensor used covers the entire visible spectrum region , having its peak optimized for the green region , so even though the colour change implies a difference in absorption of the biological compound , the photo sensor will continue to obtain a signal . meanwhile , blank and the non - complementary dna absorbs much less radiation from the light source , allowing a difference in the reading when compared to complementary dna . the difference on the reading between solutions 3 and 4 is also a proof that the system allows the quantification of the samples , in accordance to the suitable selection of a calibration algorithm . in terms of the liquid forming a drop on the surface of the sensor , the influence of the drop effect exists . this physical phenomenon is due both to the refractive index difference between the liquid and the air , and to the geometry of the drop ( convex ), which acts like a lens . despite this , the detected difference between measured values for complementary and non - 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