WO2012125122A1

WO2012125122A1 - An apparatus and method for monitoring and/or detecting the presence of at least one molecule in bodily fluid

Info

Publication number: WO2012125122A1
Application number: PCT/SG2012/000082
Authority: WO
Inventors: Kiang Wei KHO; Mingwei FAN
Original assignee: Novi Biotech Pte Ltd
Priority date: 2011-03-11
Filing date: 2012-03-12
Publication date: 2012-09-20

Abstract

An apparatus for monitoring and/or detecting the presence of at least one molecule in bodily fluid is disclosed. The apparatus comprises: an extraction unit configured to extract the bodily fluid, the extraction unit comprising a micro-needle; and at least one SERS-active member arranged with the extraction unit to allow the extracted bodily fluid to contact the at least one SERS-active member for generating SERS signals of the extracted bodily fluid, the apparatus being configured to monitor and/or detect the presence of at least one molecule in the extracted bodily fluid based on the generated SERS signals.

Description

An Apparatus and Method for Monitoring and/or Detecting the Presence of At

Least One Molecule in Bodily Fluid

Field of the invention

The present invention relates to an apparatus and method for monitoring and/or detecting the presence of at least one molecule in bodily fluid.

Background of the art

Continuous monitoring of human health or drug effects by checking blood (or other bodily fluids) for health index markers (usually proteins) or drug compounds plays an important role in the diagnosis and management of several diseases [Kazuyoshi Tsuchiya, et. al., Biomedical Microdevices, 7(4), 347-353, 2005]. In a clinical setting, measurement of the homeostatic level of bio-analytes in blood is the most common mean for health and drug monitoring, owing to the ease with which blood samples can be collected [Manju Venugopal, et. al. IEEE sensors journal, 8(1 ), 71 , 2008]. The procedure normally involves the extraction of blood (or other bodily fluids) from the patient with either a lancet or a syringe, followed by an ex-vivo analysis of the collected samples in a lab using an appropriately-chosen assay technique, e.g. Enzyme- Linked Immuno-Sorbent Assay (ELISA). It is crucial that the lag time between the sample collection and the acquisition of the final results be kept to a minimum. For instance, a rapid analysis of serum S-100B protein levels in children with head trauma can greatly assist their clinical diagnosis and the management of high-risk patients in the emergency units [Hallen M, et. al., J. Trauma, 2010, 69(2), 284-9]. As such, a real-time monitoring system for biomarkers is highly desirable. Being able to extract blood samples painlessly is also important in a blood monitoring system. For instance, in a non-clinical setting or in cases where patients have a very low pain tolerance (e.g. in children), a painless real-time monitoring could significantly improve patient compliance [Ping M. Wang, et. al., Diabetes technology & therapeutics, 7(1 ), 2005, 131 ; Klonoff D., Diabetes Care 1997, 20, 433-437]. In diabetes mellitus, the determination of the sugar level in extracted blood is extremely important for the diagnosis and effective management of the condition. However, the compliance with glucose monitoring by diabetic patients has been poor because of the pain and inconvenience of conventional blood collection using lancets [Ping M. Wang, et. al., Diabetes technology & therapeutics, 7(1 ), 2005, 131 ]. Using conventional technology, it is necessary to make between 5 to 7 measurements of glucose concentrations each day for a typical diabetic patient in order to stabilise his/her blood glucose level [Kazuyoshi Tsuchiya, et. al., Biomedical Microdevices, 7(4), 347-353, 2005]. The process of making each of these measurements is usually painful for the patient. Therefore, to improve compliance, many studies have sought to develop and test minimally invasive approaches to extract bodily fluid for continuous blood monitoring. [Marwick C, JAMA 1998, 280, 312 - 313]

RAMAN SPECTROSCOPY

Two optical processes can occur when a monochromatic light interacts with a molecule. In the first, and the dominant process, a large portion of the incident light is elastically scattered with no photon energy being absorbed - this is known as the Rayleigh scattering. A secondary optical phenomenon relates to a process in which a small amount of the incident photon energy is absorbed and results in a transition between vibrational states within the probed molecule. This process unfortunately is relatively weak and would generally lead to a subsequent "re-emission" of a photon quantum whose frequency is "shifted" from that of the incidence light. This optical effect is conventionally known as Raman scattering or Raman spectroscopy (RS), and was first observed experimentally by Raman and Krishnan in 1928 [Raman C. V., et. al., Nat. 1928, 121 , 501 ]. Fig. 1 depicts two types of Raman scattering: (i) Stokes Raman scattering whereby the Raman photon energy is less than the incident photon energy and (ii) Anti-Stokes Raman Scattering whereby the Raman photon-energy is greater than the incident photon energy.

Raman frequency shifts of a molecule are closely related to the vibrational modes - here, vibrational modes refer to the "manner" in which the molecule vibrates, which in turn is dependent upon both the molecular structures as well as the chemical nature of the molecule's immediate surroundings [Jeanmaire, D. L, et. al., J. Electroanal. Chem. 84(1 ), 1977, 1]. SURFACE ENHANCED RAMAN SPECTROSCOPY

While RS is able to provide high chemical specificities, such a feature is offset by the weak Raman scatterings in most molecules. It is estimated that only about one-millionth of the incident photons is "Raman-scattered" by a given molecule.

Such a shortcoming can be overcome by field enhancements occurring on optically-excited nano-structured metallic surfaces in a phenomenon conventionally known as Surface Enhanced Raman Spectroscopy or SERS.

SERS was first discovered by Fleischman et al in 1974 when they observed remarkably strong Raman signals for pyridine adsorbed on an electrochemically roughened silver electrode [Reischman, M., et. al. Chem. Phys. Lett. 1974, 26, 123]. However, the mechanism responsible for such an observation was not understood, and was initially attributed to the increased surface area, until 1977, where it was recognized, by two separate groups, Jeanmarie and Van Duyne, Albercht and Creighton, that the observed enhancement could be attributed both to surface plasmon resonance and to chemical effects on the metallic surfaces [Jeanmaire, D. L, et. al., J. Electroanal. Chem. 84(1 ), 1977, 1]. Ever since, enhanced Raman scattering has been reported for other compounds that have been brought into close proximity with a metallic surface (typically Silver, Gold or Copper) with nano-scale features. Generally, it is widely accepted that two mechanisms are responsible for enhancements in Raman scatterings (which can be as high as 10¹⁴ times the unenhanced signal): [Kneipp, K., et. al. Chem. Rev. 1999, 10, 2957] Electromagnetic enhancement (EM) and Chemical enhancement (CM).

Electromagnetic enhancement

The majority of the SERS effects can be accounted for by electromagnetic enhancements arising from interactions between the adsorbate analyte and the surface plasmon (SP) fields produced on the metallic nano-structured surface by the excitation laser beam. SP is the-eelleetive oscillation-of-eharges^~bound to the metal-dielectric interface brought about as a result of coupling between the light-fields and the surface charges. On a roughened metallic surface, such a coupling becomes extremely efficient, and the resultant oscillating SP fields generated are thus concomitantly amplified. As a consequence, this leads to the adsorbed analyte molecule experiencing a strong laser excitation, and in turn results in a large Raman scattering, i.e. the SERS effect.

Chemical enhancement

Chemical enhancement contributes only an order of 10- 0² to the overall enhancement, and is currently not fully understood. Nonetheless, the widely accepted mechanisms are that charge-transfers between the analyte and metallic surface form an analyte-surface species capable of coupling, resonantly, with the excitation light, thereby leading to amplified Raman scatterings. The strength of chemical enhancement is generally affected by the surface potential.

PRIOR ARTS

Conventional approaches to real-time monitoring of bio-analytes in bodily fluid based on the technique of SERS are generally ex-vivo; sensing is not performed at the instance the sample is collected. Only a handful of SERS- based in-vivo sensing has been published. An interesting example is that reported by Sulk et al. [Sulk et al., Journal of Raman Spectroscopy 30:853 [1999]], in which a SERS-active nano-structured substrate was surgically implanted subcutaneously under the skin of a rat to allow for in-vivo SERS measurements of serum glucose levels. Raman excitation and signal collection were achieved via an optical window integrated into the substrate. While the report has shown good measurement reversibility and substrate stability, the invasiveness of such a technique implies that it is highly unlikely to become a main-stream approach for real-time sensing.

A proposed alternative means for blood extraction in a minimally invasive way is through the application of a low-energy laser to create micropores in the stratum corneum as reported by Manju Venugopal, et al. [Manju Venugopal, et. al. IEEE Sensors journal, 8(1 ), 2008, 71]. The primary objective of this approach is to access the interstitial fluid (ISF) instead of blood plasma, since both fluids are thought to contain similar composition. Clinical tests involving diabetic patients have shown that the correlation between the ISF glucose concentration and blood glucose levels is as high as 0.90 in the 60 - 400 mg/dl glucose range [Gebhart S., et al. Diabetes Technol. Ther. 5, 159, 2003]. This technique has been used in the continuous monitoring of glucose [Daniloff G. Y., Diabetes Technol. Ther. 1 , 261 , 1999].

Although small to moderate sized molecules, for example glucose and ethanol, are generally found in ISF in the same proportion as in blood, larger molecules such as certain lipids or disease-related proteins are present at a much reduced concentration relative to blood [Bantle J. P., et al. J. Lab. Clin. Med. 130, 436, 1997]. Additionally, a lag time of up to 38 min between the levels of various molecules (for example, glucose and ethanol) in blood and in ISF can contribute to significant measurement errors in continuous monitoring systems, thereby reducing its clinical utility [Stout P. J., et al., Diabetes Technol. Ther. 1 , 21 , 1999]. Micro-needle systems, on the other hand, can achieve the appropriate level of invasiveness suitable for good patient compliance and for regular blood sampling. A micro-needle generally comprises biocompatible material and has an external and internal diameter of a few tens of micro-meters [Kazuyoshi Tsuchiya, et. al., Biomedical Microdevices, 7(4), 347-353, 2005]. Its small size mimics the labium of a female mosquito, providing a painless means for extracting human blood. A micro-needle can be made of Silicon (Si), Titanium (Ti) or Gold (Au). Blood extraction with a micro-needle can be achieved through either capillary forces or the application of negative pressures. An excellent example is the compact wristwatch type self-monitoring blood glucose system based on a micro-needle array .eported by Kazuyoshi Tsuchiya, et. al. [Kazuyoshi Tsuchiya, et. al., Biomedical Microdevices, 7(4), 347-353, 2005]. As compared to the ISF extraction approach, the micro-needle, due to its larger internal diameter, provides a more direct access to the blood, hence eliminating the issues of lag-times as well as diminished concentrations of large molecules, e.g. proteins.

However, thus far, a micro-needle serves merely as a conduit for blood collection, and, in itself, lacks any bio-sensing capability. Fluid collected through the needle must be directed to a separate location or platform for bio-analysis. This would result in a huge volume of blood being drawn owing to the large distance between the micro-needle system and the sensor. Furthermore, a sophisticated micro-pump system would be needed to maintain negative pressures for a sufficient period of time until a suitable amount of blood has been delivered to the sensing platform.

Summary of the invention

The present invention addresses the problems above, and in particular provides a novel and useful apparatus and method for monitoring and/or detecting the presence of at least one molecule in bodily fluid. According to a first aspect of the present invention, there is provided an apparatus for monitoring and/or detecting the presence of at least one molecule in bodily fluid, the apparatus comprising: an extraction unit configured to extract the bodily fluid, the extraction unit comprising a micro-needle; and at least one SERS-active member arranged with the extraction unit to allow the extracted bodily fluid to contact the at least one SERS-active member for generating SERS signals of the extracted bodily fluid, the apparatus being configured to monitor and/or detect the presence of at least one molecule in the extracted bodily fluid based on the generated SERS signals.

According to another aspect of the present invention, there is provided a method for monitoring and/or detecting the presence of at least one molecule in bodily fluid, the method comprising: contacting the bodily fluid with the at least one SERS-active member of the apparatus according to any aspect of the present invention to generate SERS signals of the bodily fluid, the bodily fluid being comprised in the micro-needle of the apparatus prior to the contact; and monitoring and/or detecting the presence of at least one molecule in the bodily fluid based on the generated SERS signals of the bodily fluid. According to a third aspect of the present invention, there is provided a system for monitoring and/or detecting the presence of at least one molecule in bodily fluid, the system comprising: an apparatus for monitoring and/or detecting the presence of at least one molecule in bodily fluid according to any aspect of the present invention; and an optical unit configured to provide signals to the apparatus for generating the SERS signals of the extracted bodily fluid and further configured to collect the generated SERS signals of the extracted bodily fluid.

According to one aspect, the present invention provides an apparatus for monitoring and/or detecting the presence of at least one molecule in bodily fluid, wherein the apparatus is substantially as described according to the whole content of the present invention. According to another aspect, the present invention provides a method for monitoring and/or detecting the presence of at least one molecule in bodily fluid, wherein the method is substantially as described according to the whole content of the present invention.

According to another aspect, the present invention provides a system for monitoring and/or detecting the presence of at least one molecule in bodily fluid, wherein the system is substantially as described according to the whole content of the present invention.

According to a further aspect, the present invention may be used for diagnosis purposes.

Brief description of the figures

Embodiments of the invention will now be illustrated for the sake of example only with reference to the following drawings, in which:

Fig. 1 shows two types of Raman scattering: Stokes Raman scattering whereby the Raman photon energy is less than the incident photon energy and Anti-Stokes Raman Scattering whereby the Raman photon-energy is greater than the incident photon energy;

Fig. 2(A) shows an apparatus for monitoring and/or detecting at least one molecule in bodily fluid according to a first embodiment of the present invention and Fig. 2(B) shows an example of using the apparatus of Fig. 2(A) for monitoring and/or detecting at least one molecule in blood;

Fig. 3 shows an apparatus for monitoring and/or detecting at least one molecule in bodily fluid according to a second embodiment of the present invention;

Fig. 4(A) shows an apparatus for monitoring and/or detecting at least one molecule in bodily fluid according to a third embodiment of the present invention and Fig. 4(B) shows an example of using the apparatus of Fig. 4(A) for monitoring and/or detecting at least one molecule in blood plasma; Fig. 5(A) shows an apparatus for monitoring and/or detecting at least one molecule in bodily fluid according to a fourth embodiment of the present invention and Fig. 5(B) shows an example of using the apparatus of Fig. 5(A) for monitoring and/or detecting at least one molecule in blood plasma;

Fig. 6 shows a variation of the apparatus of Fig. 2(A);

Fig. 7 shows an optical-fiber collection system comprising the apparatus of Fig. 5;

Fig. 8(A) shows a method for fabricating an Au-nanoparticles decorated Au-surface (Au-NP-Au) and Fig. 8(B) shows the Au-NP-Au obtained from the method of Fig. 8(A);

Fig. 9 shows a SERS spectrum of 4-MBA obtained using an Au-NP-Au prepared using the method of Fig. 8(A) with a first fabrication condition;

Fig. 10 shows a SERS spectrum of 4-MBA obtained using an Au-NP-Au prepared using the method of Fig. 8(A) with a second fabrication condition;

Fig. 1 1 shows a SERS spectrum of 4-MBA obtained using an Au-NP-Au prepared using the method of Fig. 8(A) with a third fabrication condition;

Fig. 12(A) shows a method for fabricating an Au-nanoparticles decorated micro-needle (Au-NP-MN) and Fig. 12(B) shows the Au-NP-MN obtained from the method of Fig. 12(A);

Fig. 13 shows an un-enhanced Raman spectrum of 10uM Crystal-Violet on a quartz cover slip;

Fig. 14 shows an enhanced Raman spectrum of 10uM Crystal-Violet acquired from a hollow core of an Au-NP-MN at an excitation wavelength of 785nm whereby the Au-NP-MN is prepared using the method of Fig. 2; and Fig. 15 shows an experimental setup comprising an Au-NP-MN fabricated using the method of Fig. 12.

Detailed description of the invention Embodiments of the current invention are concerned with merging a minimally- invasive micro-needle extraction system with an optical-based bio-sensing capability to form a convenient apparatus for monitoring and/or detecting the presence of molecule(s) in bodily fluid such as blood. More specifically, embodiments of the invention seek to combine the technique of Surface- Enhanced Raman Spectroscopy (SERS) with the micro-needle technology in order to achieve real-time in-vivo bio-sensing of bodily fluid (e.g. blood) in a minimally invasive fashion. By "in-vivo", it is meant that the SERS measurements may be performed while the micro-needle is embedded in the skin of a patient and the bodily fluid need not be removed from the apparatus in the embodiments of the present invention for performing the measurements. Embodiments of the current invention aim to render the micro-needle extraction system dual-functional: to extract bodily fluid (e.g. blood) as well as to carry out bio-sensing. This achieves a much simpler and more compact bodily fluid monitoring system. Thus, the length through which the bodily fluid has to travel before it reaches the sensing platform in the embodiments of the present invention is generally lower than that in the prior arts. More specifically, in the embodiments of the present invention, the length through which the bodily fluid has to travel before it reaches the SERS sensing platform ranges from 700 m to 3.8mm. Furthermore, embodiments of the current invention relate to methods for the painless in-vivo bio-analysis of blood with the technique of SERS.

Fig. 2(A) illustrates an apparatus 200 for monitoring and/or detecting at least one molecule in bodily fluid. The apparatus 200 comprises a planar platform 202 with a first side comprising a hollow micro-needle 204 and a second side comprising SERS-active members in the form of metallic nano-structures 206. The micro-needle 204 serves as a part of an extraction unit of the apparatus 200 whereby the extraction unit is configured to extract the bodily fluid. Note that a single SERS-active member may be sufficient. However, this is not preferable as the obtained SERS signals are likely to be weak if the apparatus 200 comprises only a single SERS-active member. Furthermore, both the micro-needle 204 and the platform 202 may be made of the same biocompatible material, such as Si or a bio-polymer for example, Polymethylmethacrylate (PMMA). Fig. 2(B) illustrates an example of using the apparatus 200 for monitoring and/or detecting at least one molecule in blood 210. The micro-needle 204 is preferably stiff and sturdy to prevent collapsing or bending when penetrating the skin. As shown in Fig. 2(B), the micro-needle 204 may be used to penetrate the skin 208 to allow blood 210 to flow through the hollow core of the micro-needle 204 and reach the nano-structures 206 on the first side of the platform 202. The blood flow may be driven by capillary forces or by the application of a negative pressure to the interior of the hollow needle 204. The nanostructures 206 are arranged with the micro-needle 204 to allow the extracted blood 210 to contact them. As the extracted blood 210 contacts the nano-structures 206, adsorption of any serum analyte onto the nanostructures 206 triggers SERS-activities. This generates SERS signals (or spectra) of the extracted blood 210. SERS signals or spectra derived using the nano-structures 206 in this manner would thus carry specific chemical information relating to the composition of the extracted blood 210.

Fig. 3 illustrates an apparatus 300 for monitoring and/or detecting at least one molecule in bodily fluid. The apparatus 300 is similar to the apparatus 200 of Fig. 2(A) and thus, the same parts will have the same reference numerals, with addition of prime. In the second embodiment as shown in Fig. 3, SERS-active nano-structures 212 are arranged along the interior of the hollow micro-needle 204' (which forms a channel of the micro-needle 204' through which the bodily fluid may be extracted). The SERS-active nano-structures 212 may be attached to the interior of the micro-needle 204'. In this case, the SERS signals may become immediately detectable as soon as the bodily fluid is drawn into the channel of the micro-needle 204'. In this embodiment, less bodily fluid (e.g. blood) needs to be extracted to produce detectable SERS signals since the bodily fluid does not have to travel the entire length of the channel of the microneedle 204' before reaching the SERS-active nano-structures 212 (unlike in the first embodiment shown in Fig. 2(A)).

Fig. 4(A) illustrates an apparatus 400 for monitoring and/or detecting at least one molecule in bodily fluid. The apparatus 400 is also similar to the apparatus 200 of Fig. 2(A) and thus, the same parts will have the same reference numerals, with addition of double prime. The apparatus 400 as shown in Fig. 4(A) comprises SERS-active nano-structures 214 and a semi-permeable membrane 216 arranged with the platform 202" comprising the micro-needle 204". The nano-structures 214 may be attached to the membrane 216 which may in turn be attached to the platform 202". The membrane 216 forms part of the extraction unit of the apparatus 400 and may comprise pores. In one example, each pore of the membrane 216 has a sub-10 μιη diameter. Fig. 4(B) illustrates an example of using the apparatus 400 for monitoring and/or detecting at least one molecule in blood plasma. As shown in Fig. 4(B), the membrane 216 serves to allow only selected components (in this case, blood plasma 218) of the extracted blood to reach and contact the SERS-active nano- structures 214. Other components of the extracted blood such as the red blood cells (RBCs) 220, whose diameters may be 5 - 6 μιη, are usually unable to diffuse through the membrane 216 (see Fig. 4(B)). In this embodiment, the amount of compounding and interfering SERS/Raman signals arising from the RBCs may be reduced.

Fig. 5(A) illustrates an apparatus 500 according to a fourth embodiment of the current invention. The apparatus 500 serves to monitor and/or detect at least one molecule in bodily fluid. The apparatus 500 is also similar to the apparatus 200 of Fig. 2(A) and thus, the same parts will have the same reference numerals, with addition of triple prime. In the fourth embodiment as depicted in Fig. 5(A), the hollow micro-needle 204"' is filled with a semi-permeable material in the form of a material comprising nano-scopic pores 222. The porous material forms part of the extraction unit of the apparatus 500. The porous material is further decorated with metallic nano-structures 224 to render it SERS-active. Such a porous structure 221 comprising the porous material and the metallic nano-structures 224 may be referred to as a SERS-active mesh (SERS-M). The SERS-M serves two functions (see Fig. 5(B)): 1 ) It serves to hold the nano-structures 224 necessary for invoking SERS activities; 2) It serves as a sieve for filtering out components such as RBCs 226 for reducing RBC-related compounding and interfering SERS/Raman signals. Owing to the large sampling volume provided by the three-dimensional (3D) structure of the SERS-M (formed by the integration of the porous material with the nano- structures 224), larger overall-Raman enhancement may be achieved using the fourth embodiment depicted in Fig. 5(A) as compared to the first and second embodiments depicted in Figs. 2(A) and 3.

Fig. 6 illustrates a variation of the apparatus 200. As shown in Fig. 6, the apparatus 200 (which may be referred to as a SERS-active micro-needle system) is rendered bio-specific by coating the SERS-active nano-structures 206 with a molecular layer of bio-analyte recognising agents 228. In one example, the molecular layer 228 comprises a partition layer that preferentially and reversibly adsorbs glucose. In this example, the SERS-active micro-needle system may be used for real-time glucose monitoring. It is to be understood that the diagram shown in Fig. 6 is merely for illustration purposes. The same concept may be applied (in other words, a similar variation may be made) to the apparatus 300, 400 and 500 depicted in Figs. 3, 4 and 5 respectively.

Fig. 7 illustrates an optical-fiber collection system 700 comprising the apparatus 500 shown in Fig. 5. This optical-fiber collection system 700 serves as a system for monitoring and/or detecting at least one molecule in bodily fluid. The optical- fiber collection system 700 further comprises an optical unit configured to provide signals to the apparatus 500 for generating the SERS signals and further configured to collect the generated SERS signals. As shown in Fig. 7, the optical unit may comprise 1 ) an optical fiber 230 for laser delivery and signal collection and 2) a focusing mechanism 232 to focus excitation beam 234 as well as to collect returned SERS signals 236 from the SERS-active nano- structures 224. A potential application of this particular embodiment is in a portable blood monitoring system. Note that the apparatus 500 in Fig. 7 may be replaced by any of the apparatus 200, 300 or 400. EXPERIMENTAL DATA

Fabrication of SERS-active Au-nanoparticles decorated Au-Surface (Au- NP-Au)

As described above, in the embodiments of the current invention, the SERS- active micro-needle system (which may be in the form of apparatus 200, 300, 400, 500 or variations thereof) comprises at least one SERS-active member in the form of sub-wavelength metallic nano-structures (SMNS) 206, 212, 214, 224 capable of undergoing plasmonic activities upon light excitation. For example, the apparatus 200 is rendered SERS-active through the decoration of nano- structures 206 on the planar side 202 (see Fig. 2) whereas the apparatus 300 is rendered SERS-active through the decorations of nano-structures 212 on the inner wall of the hollow needle 204' (see Fig. 3).

Experiments were performed to demonstrate the feasibility of the above- mentioned nano-structures decorations. More specifically, the experiments aim to identify fabrication conditions under which SMNS may be secured to a solid surface, while, at the same time, possess the necessary surface-geometry appropriate for eliciting SERS activities. Under these fabrication conditions, the SMNS may be permanently attached to the solid surface. The experiments are further described below.

In the experiments, 40-nm gold nanoparticles (i.e. Au-nanoparticles or Au-NPs) were used as the SMNS and were attached to a planar silicon (Si) substrate (the solid surface) coated with an Au layer to form an Au-nanoparticles decorated Au-surface (Au-NP-Au). Note that although Si was used in these experiments, the general concept of the technique as described below is equally applicable to the surfaces of other solid materials, including, but not limited to, Polymethylmethacrylate (PMMA) and stainless steel, of which some micro-needle platforms can be made. Fig. 8(A) shows a method (or fabrication procedure) for fabricating the Au-NP- Au in the experiments. Note that the method shown in Fig. 8(A) serves simply as an example. Any other method suitable for fabricating the Au-NP-Au may also be used.

In the method shown in Fig. 8(A), the solid surface of interest (Si) 802 is first pre-coated with a thin layer of metal 804 using e-beam evaporation. Preferably, the metal selected for use as the metal surface 804 is one to which propanedithiol can bind with high affinity. An example of such a metal is gold (Au) which was used as the metal surface 804 in the experiments.

Next, a functionalization process is performed using a self-assembled (SAM) layer of propanedithiol 808 to render the metal surface 804 "sticky" (in other words, adhesive) to the Au-NPs 806 which serve as the SMNS. In the experiments, functionalization of the Au surface 804 was achieved by soaking the Au-Si (804 together with 802) in 1 mM propanedithiol solution prepared in ethanol, for 1 hour. Using propanedithiol solutions with higher concentrations (i.e. propanedithiol solutions equal to or above 1 mM) is preferable as it achieves the saturation of the Au surface 804 with the propanedithiol molecules 808 and allows the immobilized propanedithiol molecules 808 to assume a "standing" configuration (see Fig. 8(B)). This "standing" configuration in turn allows the exposed thiol terminals further away from the Au surface 804 (see for example 810 in Fig 8(B)) to serve as anchoring points for the Au-NPs 806. The functionalized Au-surface 804 is then washed thoroughly with ethanol to eliminate un-bound propanedithiol and the treated Au-surface 804 is then incubated in a solution of Au-NPs 806 overnight for about 12 to 15 hours. This step facilitates attachment of the Au-NPs 806 onto the Au-surface 804 via the propanedithiol layer 808 to form the Au-NP-Au 812 (as shown in the last step of Fig. 8(A) and in Fig. 8(B)). SERS-efficacy of Au-NP-Au prepared from colloidal Au-NPs solutions of different ionic-strengths

As mentioned above, the experiments aim to identify fabrication conditions under which SMNS may be secured to a solid surface, while, at the same time, possess the necessary surface-geometry appropriate for eliciting SERS activities. More specifically, the experiments aim to identify fabrication conditions upon which a self-assembled layer of Au-NPs from a colloidal solution forms a highly SERS-active surface topology on an Au surface. Using 4-Mercaptobenzoic acid (4-MBA) molecules as the test molecules, a total of three fabrication conditions were tested in the experiments.

Fabrication condition #1

In the first experiment, the Au-NP-Au 812 was prepared by incubating the functionalized Au-surface 804 in a colloidal solution of 40-nm Au-NPs 806 dispersed in water. Fig. 9 shows the SERS spectrum of the 4-MBA obtained using the Au-NP-Au 812 prepared in this experiment.

Fabrication condition #2

In the second experiment, the Au-NP-Au 812 was prepared by incubating the functionalized Au-surface 804 in a colloidal solution of 40-nm Au-NPs 806 dispersed in 100^χ diluted Phosphate Buffered Saline (PBS). Fig. 10 shows the SERS spectrum of the 4-MBA obtained using the Au-NP-Au 812 prepared in this experiment.

Fabrication condition #3

In the third experiment, the Au-NP-Au 812 was prepared by incubating the functionalized Au-surface 804 in a colloidal solution of 40-nm Au-NPs 806 dispersed in 25^χ diluted PBS. Fig. 1 1 shows the SERS spectrum of the 4-MBA obtained using the Au-NP-Au 812 prepared in this experiment. From Figs. 9 - 1 1 , it can be seen that the SERS spectrum of the 4-MBA comprises a visible peak at 1 180cm^"1 regardless of the fabrication condition. In other words, all the fabrication conditions in the experiments allow the Au-NPs 806 to be successfully secured to the Au solid surface 804 and allow the elicitation of SERS activities. Based on the peak intensity at 1 180 cm^"1, it can also be seen from Figs. 9 - 1 1 that fabrication condition #2 is preferable as the Au-NP-Au 812 produced under this fabrication condition exhibits the strongest SERS activity. Fabrication of SERS-active Au-nanoparticles decorated micro-needle (Au- NP-MN)

In a further experiment, 40-nm Au-NPs (the SMNS) were attached to the inner wall of a hollow-core stainless steel micro-needle (SS-MN) to form an Au- nanoparticles decorated micro-needle (Au-NP-MN). Note that although a SS- MN was used, the general concept of the current technique is equally applicable to micro-needles made of other materials, including, but not limited to, Polymethylmethacrylate (PMMA) and Si. The Au-NP-MN in this further experiment is similar to the apparatus 200 and 300 as shown in Figs. 2 and 3, except that it comprises SERS-active members in the form of Au-NPs on both a side of its planar platform (similar to the apparatus 200) and along the interior of the hollow micro-needle (similar to the apparatus 300).

Fig. 12 depicts a method (or fabrication procedure) for fabricating the Au-NP- MN in this further experiment. This method may also be used in a similar manner for fabricating the apparatus 200, 300, 400, 500 and variations thereof. Note that the method shown in Fig. 12 serves simply as an example. Any other method suitable for fabricating a SERS-active micro-needle in the form of the Au-NP-MN may also be used. In the fabrication procedure shown in Fig. 12, the extraction unit comprising the micro-needle 1202 is first pre-coated with a thin layer of metal 1204 by sputtering on the planar side. The metal to be used as the metal layer 1204 is preferably selected such that propanedithiol can bind to it with high affinity. An example of such a metal is gold (Au) which was used as the metal layer 1204 in the further experiment.

Next, a functionalization process is performed on the metal layer 1204 using a self-assembled (SAM) layer of propanedithiol 1206 to render the metal layer 1204 "sticky" (in other words, adhesive) to the Au-NPs (the SMNS) 1208. In the further experiment, functionalization of the Au-surface 1204 was achieved by soaking the Au-coated micro-needle i.e. Au-MN (1204 together with 202) in 1 mM propanedithiol solution prepared in ethanol, for 1 hour. Using propanedithiol solutions with higher concentrations (i.e. propanedithiol solutions with concentrations equal to or above 1 mM) is preferable as it achieves the saturation of the Au surface 1204 with the propanedithiol molecules 1206 and allows the immobilized propanedithiol molecules 1206 to assume a "standing" configuration similar to that shown in Fig. 8(B). This "standing" configuration in turn allows the exposed thiol terminals further away from the Au surface 1204 (see for example 8 0 in Fig. 8(B)) to serve as anchoring points for the Au-NPs 1208.

The functionalized Au-MN is then washed thoroughly with ethanol to eliminate un-bound propanedithiol. Next, the functionalized Au-surface 1204 is treated with a colloidal solution of Au-NPs 1208 in accordance to fabrication condition #2 as mentioned above. This facilitates attachment of the Au-NPs 1208 onto the Au-surface 1204 to produce a highly SERS-active surface topology. An Au-NP- MN 1210 is thus formed. SERS-efficacy of Au-NP-MN

Crystal-Violet was used as the test sample to test the SERS-efficacy of the Au- NP-MN 1210 in this further experiment.

Fig. 13 shows an un-enhanced Raman spectrum of 10uM Crystal-Violet on a quartz cover slip whereas Fig. 14 shows an enhanced Raman spectrum of 10uM Crystal-Violet acquired from the hollow core of an Au-NP-MN 1210 (fabricated using the method of Fig 12) at an excitation wavelength of 785 nm.

The spectrum of the Crystal Violet comprising the Crystal Violet peaks is usually not visible with un-enhanced Raman spectroscopy but is usually visible with SERS due to the enhancement of the signals of the Crystal Violet with SERS. From Figs. 13 and 14, it can be seen that the SERS spectrum acquired from the hollow core of the Au-NP-MN 1210 comprises a visible peak at 1400cm^"1 and two visible peaks at 1600cm^"1 (corresponding to the spectrum of the Crystal Violet) whereas these peaks are not present in Fig. 13. In other words, it can be seen from Figs. 13 and 14 that the Au-NPs 1208 were successfully secured to the core of the Au-MN (1204 together with 1202) and SERS activities were elicited.

Fig. 15 shows the experimental setup used in this further experiment. As shown in Fig. 15, the experimental setup comprises an objective lens 1502 for focusing the excitation laser 1504 into, as well as to collect SERS signals 1506 from the Au-NP-MN 1210. A droplet of CV solution is shown as the test sample in Fig. 15 and may be replaced by any other test sample.

Claims

1 . An apparatus for monitoring and/or detecting the presence of at least one molecule in bodily fluid, the apparatus comprising:

an extraction unit configured to extract the bodily fluid, the extraction unit comprising a micro-needle; and

at least one SERS-active member arranged with the extraction unit to allow the extracted bodily fluid to contact the at least one SERS-active member for generating SERS signals of the extracted bodily fluid, the apparatus being configured to monitor and/or detect the presence of at least one molecule in the extracted bodily fluid based on the generated SERS signals.

2. An apparatus according to claim 1 , wherein the micro-needle comprises a channel through which the extraction unit is configured to extract the bodily fluid and the at least one SERS-active member is arranged along the channel.

3. An apparatus according to claim 1 , wherein the extraction unit further comprises a semi-permeable membrane arranged with the at least one SERS- active member to allow only selected components of the extracted bodily fluid to contact the at least one SERS-active member.

4. An apparatus according to claim 2, wherein the extraction unit further comprises a semi-permeable material arranged with the at least one SERS- active member along the channel to allow only selected components of the extracted bodily fluid to contact the at least one SERS-active member.

5. An apparatus according to claim 4, wherein the at least one SERS-active member is integrated with the semi-permeable material to form a semipermeable three dimensional structure.

6. An apparatus according to any of the preceding claims, wherein the at least one SERS-active member is coated with at least one bio-analyte recognising agent.

7. A system for monitoring and/or detecting the presence of at least one molecule in bodily fluid, the system comprising:

an apparatus for monitoring and/or detecting the presence of at least one molecule in bodily fluid according to any of the preceding claims; and

an optical unit configured to provide signals to the apparatus for generating the SERS signals of the extracted bodily fluid and further configured to collect the generated SERS signals of the extracted bodily fluid.

8. A method for monitoring and/or detecting the presence of at least one molecule in bodily fluid, the method comprising:

contacting the bodily fluid with the at least one SERS-active member of the apparatus according to any of claims 1 - 6 to generate SERS signals of the bodily fluid, the bodily fluid being comprised in the micro-needle of the apparatus prior to the contact; and

monitoring and/or detecting the presence of at least one molecule in the bodily fluid based on the generated SERS signals of the bodily fluid.

9. An apparatus for monitoring and/or detecting the presence of at least one molecule in bodily fluid, wherein the apparatus is substantially as described according to the whole content of the present invention.

10. A system for monitoring and/or detecting the presence of at least one molecule in bodily fluid, wherein the system is substantially as described according to the whole content of the present invention.

1 1 . A method for monitoring and/or detecting the presence of at least one molecule in bodily fluid, wherein the method is substantially as described according to the whole content of the present invention.

12. A method for fabricating the apparatus according to any of claims 1 - 6, the method comprising:

pre-coating the extraction unit with a metal layer;

functionalizing the metal layer to allow the metal layer to be adhesive to the at least one SERS-active member; and

treating the functionalized metal layer with a solution comprising the at least one SERS-active member.

13. A method according to claim 12, wherein the metal layer comprises gold.

14. A method according to claim 12 or 13, wherein the metal layer is functionalized using a propanedithiol solution with a concentration equal to or above 1 mM.

15. A method according to any of claims 12 - 14, wherein the at least one SERS-active member comprises 40-nm gold nanoparticles and the functionalized metal layer is treated with a solution of 40-nm gold nanoparticles dispersed in 100x diluted Phosphate Buffered Saline.