This application claims priority to and the benefit of Taiwan Patent Application No. 106101977, filed on Jan. 19, 2017, in the Taiwan Intellectual Property Office, the entire content of which is incorporated herein by reference.
The present disclosure relates to a nanoaggregate-embedded bead (NAEB) for detecting a bioparticle, in particular, to a magnetic NAEB, a manufacturing method thereof and a bioparticle detection method using the same.
Food-borne diseases have received a lot of attention nowadays by World Health Organization (WHO), which not only affect people's health, but also relate to the cost of medical resources. Main pathogenic bacteria of food-borne diseases are bioparticles of Salmonella and E. coli. The conventional detection methods, such as enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR), generally require a pre-enrichment or selective enrichment step first and then perform the biotype test or serotype identification. The conventional detection methods are time-consuming and cumbersome, so scientists are still committed to the development of rapid screening methods for pathogens, and Salmonella is one of the important subject matters. Thus, a NAEB Raman tag is proposed to detect the bioparticle, and the bioparticle comprises bacteria, virus, or cell, such as Salmonella or circulating tumor cell.
The NAEB Raman tag is the combination of the molecules with strong Raman scattering characteristics (named Raman reporter molecules) and the metal nanoparticles. The adsorbed molecule at the surface of the metal nanoparticles is excited by a field generated by the incident light and the Raman scattering light. When there are resonances between the excitation and scattered fields and surface plasmons, electromagnetic field enhancement at the nanoparticle surface occurs and leads to enhancement of the Raman scattering signal. Through the plasmon resonance generated by metal nanoparticles, the surface-enhanced Raman scattering (SERS) spectrum of the Raman reporter molecules can be obtained. Furthermore, a hot spot is generated at the junction between the metal nanoparticles in the NAEB Raman tag, thus the field at the hot spot is enhanced dramatically, and the Raman signal produced by the adsorbed molecule at the hot spot is also increased dramatically. However, when using the NAEB Raman tag to detect the bioparticles without the background by the NAEB Raman tag itself, a separation step with the addition of magnetic nanoparticles is in general performed. Such an additional step makes the experimental procedures more cumbersome.
Additionally, the antibody molecules on the NAEB Raman tag and the magnetic nanoparticles will compete for the binding sites of the epitope group of the same type, and such binding sites may locate in close regions. Thus, the steric hindrance for binding may happen, and the number of available binding sites may decrease. Or alternatively, two different antibodies for two different epitope groups on the bioparticle may be separately conjugated on the NAEB Raman tag and the magnetic nanoparticles. However, some bioparticles may have only one epitope group.
To solve at least one of the above technical problems, one objective of the present disclosure is to provide a magnetic NAEB, a manufacturing method thereof and a bioparticle detection method using the same.
According to at least one objective of the present disclosure, a magnetic NAEB is provided. The magnetic NAEB has a protective nanoshell, noble metal nanoparticles, Raman reporter molecules and at least one magnetic nanoparticle. The protective nanoshell is for example made of silica, polymer, or metal oxide such as titania and zirconia, and covers the noble metal nanoparticles, the Raman reporter molecules and the at least one magnetic nanoparticle. The noble metal nanoparticles can be gold nanoparticles or silver nanoparticles. The noble metal nanoparticles, the Raman reporter molecules, and the at least one magnetic nanoparticle form a magnetic nanoaggregate.
According to at least one objective of the present disclosure, a manufacturing method of the magnetic NAEB is provided. Firstly, noble metal nanoparticles, Raman reporter molecules and magnetic nanoparticles form magnetic nanoaggregates. Then, protective nanoshells cover the magnetic nanoaggregates.
According to one objective of the present disclosure, a bioparticle detection method using magnetic NAEBs is provided. Firstly, provide the magnetic NAEBs as mentioned above. Then a targeting molecule, such as antibody, aptamer, DNA, glycan, or chemical group, is conjugated on the surface of the magnetic NAEBs to provide the functionalized magnetic NAEBs. Next, make the magnetic NAEBs bind to one or more bioparticles. Utilize a magnetic component to attract the bioparticles bound with the magnetic NAEBs and the unbound magnetic NAEBs. Keep the portion of bioparticles bound with the magnetic NAEBs and the unbound magnetic NAEBs that has been attracted by the magnetic component. Utilize a filtering membrane to filter out the unbound magnetic NAEBs while the bioparticles bound with the magnetic NAEBs are kept on the filtering membrane. Utilize a Raman microscope to observe a SERS spectrum of the bioparticles bound with magnetic NAEBs which are left on the filtering membrane, so as to detect the bioparticles.
Accordingly, the magnetic NAEB, the manufacturing method thereof and a bioparticle detection method using the same may have one or more advantages as follows.
(1) The manufacturing cost of the magnetic NAEB is not high, thus having the low cost advantage.
(2) The magnetic NAEB consists of at least one magnetic nanoparticle, thus omitting the cumbersome experimental steps, saving the long pre-treatment time, further achieving the fast separation and enrichment, and having advantages of convenient operation and fast detection.
(3) The magnetic NAEB can be modified to have the targeting molecule being unique to the bioparticle to be detected, so as to perform the qualitative and quantitative analysis of the bioparticle to be detected, and have the advantages of the high accuracy and the unique identification.
The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
An exemplary embodiment of the present disclosure provides a magnetic NAEB, wherein at least one magnetic nanoparticle is included in a magnetic nanoaggregate with the favorable SERS characteristic, and a protective nanoshell, which is made of silica, polymer, or metal oxide such as titania and zirconia, covers the nanoparticles, thereby preventing the magnetic nanoaggregate from disintegration. Overall, the magnetic NAEB provided by the exemplary embodiment of the present disclosure has the technical results of fast magnetic separation and pre-concentration. Preferably, a chemical modification process can be performed to conjugate one or more targeting molecules (such as antibody, aptamer, DNA, glycan, or chemical group) on the outer wall of the protective nanoshell, wherein the targeting molecules correspond to the surface functionality of the bioparticles to be detected. Thus, the magnetic NAEB has characteristics of biostability, biocompatibility and unique binding.
Additionally, in the exemplary embodiment of the present disclosure, the filtering membrane can filter bioparticles bound with the magnetic NAEBs, so as to leave the filtered bioparticles on the filtering membrane. Then, a Raman microscope is used to observe a SERS spectrum of the magnetic NAEBs which are bound to the bioparticles and left on the filtering membrane, so as to detect the bioparticles. Furthermore, another one exemplary embodiment of the present disclosure provides a manufacturing method of magnetic NAEBs.
Firstly, referring to
In addition, the targeting molecule 15 is conjugated on the outer wall of the protective nanoshell 11 by performing a chemical modification, wherein the type of the targeting molecule 15 corresponds to the type of the surface functionality on the bioparticle where the bound magnetic NAEB 1 is dedicated for detection. The targeting molecule 15 can be antibody, aptamer, DNA, glycan, or chemical group, and the present disclosure is not limited thereto. It is noted that the targeting molecule 15 is not the essential component of the magnetic NAEB 1 provided by the exemplary embodiment of the present disclosure, and that is in some applications, the magnetic NAEB 1 does not has the targeting molecule 15.
In the exemplary embodiment of the present disclosure, through the control of the concentration of the Raman reporter molecules 13 in the solution and the pH value of the solution, the noble metal nanoparticles 12 are attracted by the Raman reporter molecules 13 to aggregate, wherein the number of the noble metal nanoparticles 12 is about 3-5, the noble metal nanoparticles 12 have an average diameter of about 5 nm-40 nm, for example 13 nm, and the present disclosure does not limit the number and the diameter of the noble metal nanoparticles 12 in NAEB. Moreover, the Raman reporter molecules 13 can be selected from at least one of Safranin O, 5-TRITC, 5(6)XRITC, MGITC and EV, and the present disclosure is not limited thereto.
In another one implementation, the magnetic nanoparticles 14 have an average diameter of 2 nm-60 nm, for example 30 nm, and the magnetic nanoparticles 14 is a nanomagnetite with an amine group (—NH2), for example a nanomonocrystalline Fe3O4, and the present disclosure does not limit the type, number, diameter and functional group of the magnetic nanoparticles 14. Since the Raman reporter molecules 13 have amine, isothiocyanate, or thiol group, and the magnetic nanoparticles 14 have the amine group, thus the attraction among the noble metal nanoparticles 12, the Raman reporter molecules 13 and the magnetic nanoparticles 14 form the magnetic nanoaggregate.
Next, referring to
1 g HAuCl4 is diluted with 1 l water, so as to produce a 2.62 mM aqueous solution of HAuCl4. Next, 0.01 g C6H9Na3O9 is dissolved in 1 ml water, so as to produce a 1% (wt) aqueous solution of C6H9Na3O9. Then, 5 ml of the 2.62 mM HAuCl4 solution is taken out and added in the double neck round bottom flask with 40 ml water. The double neck round bottom flask is put in the reflow device, and a magnet bar is added to stir and heat the HAuCl4 solution until the HAuCl4 solution boils. Next, under the boiling condition of the HAuCl4 solution, 1 ml of the 1% (wt) C6H9Na3O9 solution is quickly added in the double neck round bottom flask, and then the resulting solution in the double neck round bottom flask is further stirred and heated for 15 minutes. Next, the solution in the double neck round bottom flask is cooled down to room temperature, and then is stored in a 4 Celsius degree icebox. Thus, the gold colloid solution is ready to be used in subsequent experiments.
Next, at step S21, an aqueous solution of the Raman reporter molecules 13 is added into the gold colloid solution, and the pH value of the gold colloid solution is adjusted, so as to make the Raman reporter molecules 13 attract to the noble metal nanoparticles 12 to generate the gold nanoaggregate. The Raman reporter molecules 13 can be Safranin O, and the details are illustrated as follows.
0.2 g NaOH is added in 50 ml water to prepare an aqueous solution of 0.1 M NaOH. Then, the 10−4 M Safranin O solution is prepared. Next, 4 ml gold colloid solution is taken out, and the 0.1 M NaOH solution is used to adjust the pH value of the gold colloid solution, wherein the pH value is adjusted to 10. Then, 30 μl of 10−4 M Safranin O solution is taken out and added in the gold colloid solution while it is rapidly stirred, and then the resulting solution is allowed to stand for 15 minutes, so as to obtain the solution of dye-induced gold nanoaggregates based on the attraction between the Raman reporter molecules 13 and the noble metal nanoparticles 12.
Next, at step S22, an aqueous solution of the magnetic nanoparticles 14 is added in the solution generated by step S21, and since one or more magnetic nanoparticles 14 can also bind to the noble metal nanoparticles 12, the noble metal nanoparticles 12, the Raman reporter molecules 13 and one or more magnetic nanoparticles 14 form the magnetic nanoaggregate. One implementation of step S22 is illustrated as follows.
The magnetic nanoparticles 14 are nano Fe3O4, for example. Firstly, the aqueous solution of nano Fe3O4 is pre-processed. The pre-processing comprises steps as follows, 120 μl aqueous solution of nano Fe3O4 with amine group is put in the centrifuge tube, a magnetic component MAG is disposed outside and below the centrifuge tube to separate the upper liquid layer and the lower liquid layer, the upper liquid layer is removed, and 120 μl water is added in the remaining lower liquid layer, so as to prepare the stock solution of nano Fe3O4. Next, 30 μl of nano Fe3O4 solution (it has 8 mg Fe per μl, and the Fe3O4 nanoparticles have an average diameter of about 30 nm) is taken out and added in the solution generated by step 21 under rapid stirring, and then the solution is allowed to stand for 15 minutes, so as to obtain the solution of magnetic nanoaggregates.
Next, at step S23, MPTMS is added in the solution generated by step S22, such that the surface of the magnetic nanoaggregates have the —Si(OCH3)3 groups. One implementation of step S23 is illustrated as follows.
1.9 μl MPTMS is taken out and dissolved in 400 μl EtOH, so as to prepare the 0.024 M MPTMS/EtOH solution. Then, 20 μl of the 0.024M MPTMS/EtOH solution is added in the solution generated by step 22 under rapid stirring, and the resulting solution is allowed to stand for 15 minutes.
Next at step S24, a sol-gel reaction of hydrolysis-condensation is performed on the surface of the MPTMS-modified magnetic nanoaggregates in the solution generated by steps S23. Therefore, the protective nanoshells 11, which is made of silica, cover the magnetic nanoaggregates, and the solution of the magnetic NAEBs 1′ is obtained. One implementation of step S24 is illustrated as follows.
12.5 μl TEOS is taken out and dissolved in 1200 μl ethanol, so as to prepared 0.0046M TEOS/EtOH solution. Subsequently, 15 ml ethanol, 500 μl of 33% (wt) NH4OH solution and 150 μl of 0.0046 M TEOS/EtOH solution are sequentially added in the solution generated by step S23 rapidly, the resulting solution is then sonicated for 30 minutes. Next, 150 μl of 0.0046M TEOS/EtOH solution is added in the solution again, and the resulting solution is sonicated for 30 minutes again (the step can be repeated three times, and totally 600 μl of 0.0046M TEOS/EtOH solution are added). Then, the above solution is further sonicated for 12 hours. Next, the resulting solution after sonication is put in a centrifuge tube and centrifuged for 12 minutes at 1200 rpm speed. Then, the upper liquid layer in the centrifuge tube is removed and the lower liquid layer is kept, and ethanol is used to rinse the remaining solution by centrifugation three times. Finally, pure water is used to dilute the remaining solution to 1 ml, and the 1 ml solution is stored in a cool space at room temperature. Therefore, the magnetic nanoaggregates covered by the protective nanoshells 11 made of the silica is obtained, and the aqueous solution of the magnetic NAEBs 1′ is obtained.
According to the detailed descriptions of
Next, referring to
1 ml aqueous solution finally generated in
Next, at step S32, a chemical modification process is performed on the magnetic NAEBs 1′, so as to conjugate the targeting molecules 15 on the outer walls of the protective nanoshells 11. Therefore, the functionalized magnetic NAEBs 1 are obtained. One implementation of the chemical modification at step S32 is illustrated as follows.
The 1 ml solution obtained by step S31 is separated to five parts (each is 200 μl), and 40 μl antibody solution and 20 μl EDC/NHS solution are added and dissolved in the 200 μl solution. Then, the 200 μl solution is sonicated for 2 hours at room temperature. The above antibody solution is prepared in PBS buffer to have a concentration of 5×10−6 g per ml. The above EDC/NHS solution is prepared by dissolving EDC and NHS in PBS buffer, wherein the EDC/NHS solution has 0.523 M EDC and 0.869 M NHS. Next, 500 μl of 10 mM quench buffer solution is added, and the resulting solution is allowed to react for 30 minutes at room temperature. The above quench buffer is prepared by dissolving 20 μl monoethanolamine in 1980 μl pure water to have a concentration of 10 mM. Next, PBS buffer is used to rinse the solution for 10 minutes at 10000 rps. The rinsing process is performed three times to remove the upper liquid layer. After the the upper liquid layer is removed, PBS buffer is added to make the solution to 11 ml, and thus the solution of magnetic NAEBs 1 is obtained. Preferably, the obtained solution can be preserved in a 4 Celsius degree icebox.
Next, referring to
Next, at step S42, the filtering membrane FLT is put in the centrifuge tube TC2, and through centrifugation, the one or more bound bioparticles are attracted by the magnetic component MAG and left on the filtering membrane FLT, while the unbound magnetic NAEBs 1 pass through the filtering membrane FLT. Next, at step S43, the filtering membrane FLT is taken out, and the Raman microscope RAS (such as confocal Raman microscope) is used to observe a SERS spectrum of the magnetic NAEBs 1 with bound Salmonella SC left on the filtering membrane, so as to detect the Salmonella SC.
If no Salmonella SC are bound with the magnetic NAEBs 1, all of the magnetic NAEBs 1 will pass through the filtering membrane FLT, and thus the SERS spectrum of the magnetic NAEBs 1 cannot be observed by the Raman microscope RAS. By contrast, if there are Salmonella SC bound with the magnetic NAEBs 1 and left on the filtering membrane FLT, the SERS spectrum of the one or more magnetic NAEBs 1 on the filtering membrane FLT can be observed by the Raman microscope RAS, so as determine whether the Salmonella SC exist, and further to determine the concentration of the Salmonella SC.
Next, referring to
Finally, referring to
Accordingly, the magnetic NAEB provided by the exemplary embodiment of the present disclosure can be used in the bioparticle detection method, and thus the bioparticle detection method has advantages of low cost, convenient operation, fast detection, and high accuracy.
The above-mentioned descriptions represent merely the exemplary embodiment of the present disclosure, without any intention to limit the scope of the present disclosure thereto. Various equivalent changes, alternations or modifications based on the claims of present disclosure are all consequently viewed as being embraced by the scope of the present disclosure.
Number | Date | Country | Kind |
---|---|---|---|
106101977 A | Jan 2017 | TW | national |
Number | Name | Date | Kind |
---|---|---|---|
20090130655 | Chau | May 2009 | A1 |
20110182815 | Daich | Jul 2011 | A1 |
Entry |
---|
Chon et al., “Simultaneous immunoassay for the detection of two lung cancer markers using functionalized SERS nanoprobes”, The Royal Society of Chemistry, ChemComm, 47, 2011, pp. 12515-12517. (Year: 2011). |
Number | Date | Country | |
---|---|---|---|
20180200296 A1 | Jul 2018 | US |