GB2590485A

GB2590485A - Nanoparticle conjugation

Info

Publication number: GB2590485A
Application number: GB1918898.6A
Authority: GB
Inventors: Leo Van Workum Stefan; Alexander Turek Vladimir; Dorrestijn Marko; R Klug David
Original assignee: Vidya Holdings Ltd
Current assignee: Vidya Holdings Ltd
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2021-06-30
Anticipated expiration: 2039-12-19
Also published as: GB2590485B; WO2021123806A1; GB201918898D0

Abstract

A semi-quantitative process for gauging nanoparticle conjugation efficiency. Measuring a spectral shift 20 (e.g. red shift) of the localised surface plasmon resonance (LSPR) maximum absorbance peak of an unconjugated nanoparticle 24 and a fully conjugated nanoparticle 22. Converting the spectral shift into a value of conjugation efficiency by comparing with a predetermined scale of nanoparticle coverage. Multiple layers of (the same) ligand may be applied.

Description

NANOPARTICLE CONJUGATION

This present invention relates to improvements in or relating to nanoparticles, and in particular, to a semi-quantitative process for gauging nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation.

Nanoparticle-antibody conjugates are used as optical or electrochemical markers in immunoassays or biomarkers, for example. The antibody must be immobilised onto the surface of the nanoparticle and this binding process typically occurs spontaneously. The immobilisation process may induce nanoparticle aggregation or may result in loss of bioactivity. Total bound antibodies to nanoparticles can be determined by subtracting the initial concentration of antibodies from the free protein detected in the supernatant by fluorescence.

Localised surface plasmon resonance (LSPR) consists of non-propagating excitations of the conduction electrons of nanoparticles coupled to the electromagnetic field. The surface plasmon energy, also known as the resonance frequency of oscillation, is determined by the dielectric properties of the ligand attached to the nanoparticle, the surrounding medium and by the nanoparticle size, shape and composition. LSPR is used in advanced biosensing with a high degree of precision, sensitivity and colorimetric detection. Plasmonic coupling of nanoparticles occurs when two or more plasmonic nanoparticles come into close contact with one another. This can lead to large shifts in LSPR frequencies. Large scale aggregation of nanoparticles in solution can be induced through analyte binding.

Assessing the nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation is of paramount importance in achieving consistent, reproducible results using antibody conjugated nanoparticles in immunoassays because such immunoassays measure the specific binding affinity against non-specific adsorption to the same surface. Furthermore, nanoparticles show large surface area to volume ratio and hence nanoparticles can have high levels of conjugation per unit volume. Complete characterisation of the conjugation process would be extremely challenging but it has been found that sufficient relevant insight can be obtained through monitoring the peak frequency of the localised surface plasmon resonance of the particles.

It is against the background that the present invention has arisen.

According to the present invention there is provided a semi-quantitative process for gauging nanoparticle conjugation efficiency. Additionally or alternatively, there may be provided a semi-quantitative process for gauging the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation. This process comprises the steps of: measuring a spectral shift of the localised surface plasmon resonance (LSPR) maximum absorbance of an unconjugated nanoparticle and a fully conjugated nanoparticle; and converting the spectral shift into a value of conjugation efficiency conjugation by comparison with a predetermined scale of nanoparticle coverage. Additionally or alternatively, the process of the present invention may involve the step of converting the spectral shift into a value to determine the extent of conjugation and/or the degree of nanoparticle conjugation.

Localised surface plasmon resonance (LSPR) is well suited to this application due to it being highly sensitivity to the refractive index of the surrounding medium, which causes a shift in the resonant frequency. Additionally, it is non-invasive because it looks at changes in refractive index and will therefore not interfere with the functioning of the assay. Nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation may be stated as a percentage and is an average over all nanoparficles in solution. As such it is an approximation of the per-particle conjugation. For example, 80% conjugation may mean that each particle has antibody attached to 80% of its surface or it may mean that 80% of the nanoparticles are fully conjugated and 20% are unconjugated.

A semi-quantitative process has been established in that a scale of nanoparticle coverage can be empirically established and used to gauge the nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation of a further sample.

The process of the present invention also comprises the preliminary calibration steps of: (i) measuring the wavelength of maximum absorbance of the LSPR in a spectrum before conjugation; (ii) measuring the wavelength of maximum absorbance of the LSPR in a spectrum of a fully conjugated nanoparticle; and (iii) determining the conjugation scale of nanoparticle coverage.

This calibration step may be carried out for each batch of nanoparficles or it may be carried out only when there is an expectation of need arising due to a different type of particle or different type of conjugation. The particles can vary in shape, size, polydispersity and/or composition. The conjugation can be of non-protein ligands or protein ligands with a different refractive index.

In some embodiments, conjugation scale of nanoparticle coverage in step (iii), may be determined by determining the difference in wavelength i.e. AN between the maximum absorbance of the LSPR before conjugation of the nanoparticle and after a fully conjugated nanoparticle. Due to the similarities of the refractive indices between proteins, the ratio of the maximum absorptions of unconjugated to fully conjugated nanoparticles gives a gauge of the coverage of nanoparticles, irrespective of the protein, therefore antibodies can be compared with for example BSA, the ratio of the maximum absorptions of unconjugated to fully conjugated nanoparticles can provide a gauge of the coverage of nanoparticles.

The present invention also provides a process of functionalising a nanoparticle for use in an immunoassay. This process can comprise the steps of: applying a first layer of ligands; and gauging the nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation using the semi-quantitative process disclosed above. This process can also comprise the steps of: applying a further layer of ligands; and gauging the nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation in a further layer of ligands using the semi-quantitative process disclosed above. The efficiency of the process can be optimised by assessing each layer for their nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation. Under the circumstances where the nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation of any one of the layers of ligands does not fit in the predetermined acceptable range, the functionalisation can be halted without wasting precious materials.

The process of functionalising a nanoparticle may include a layer of ligands such that the nanoparticle can comprise a homogenous population of ligands to enable tight control to be maintained over the nanoparticle conjugation.

The process of functionalising a nanoparticle may include a series of layers conjugating to the nanoparticle that can comprise more than one type of ligand, wherein each of the different ligand types will be in a separate layer.

In some embodiments, one ligand can initially coat the nanoparticle. However, if the coverage is not sufficient, the particles may be prone to aggregation/adsorption to surfaces.

To prevent this, a second treatment with a non-stick moiety could be performed for example, polymers such as polyethylene glycol or surfactants such as Tween 20 or even other proteins such as BSA.

In some embodiments, the coverage of the ligand is deliberately lowered to fine-tune the affinity of the nanoparticles to the markers, but in order to prevent other species adsorbing to the 'free' surface of the nanoparticles, some other species is used to 'block' this.

The process of functionalising a nanoparticle may include more than one type of ligand being included in at least one layer. The provision of multiple ligands in a single funcfionalisation step enables all of the ligands to access a fully unconjugated nanoparticle.

Nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation is stated as a value. Nanoparficle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation may be stated as numerical indicia, percentage, a ratio or a fraction. This value of conjugation efficiency, the extent of conjugation and/or the degree of conjugation is an average over all nanoparticles in solution. As such it is an approximation of the per-particle conjugation. For example, 80% conjugation may mean that each particle has antibody attached to 80% of its surface or it may mean that 80% of the nanoparticles are fully conjugated and 20% are unconjugated.

In some embodiments, the approximation of the per-particle conjugation may be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

The ligand may be an antibody. Antibodies are used as a ligand due to their high biocompatibility. The conjugation of nanoparticles with antibodies combines the properties of the nanoparticles themselves with the specific and selective recognition ability of the antibodies as capture agents for use in immunoassays. The ligand can also be a nucleic acid, protein, polymer, small molecule or ion.

Nucleic acids can have a natural affinity or selectivity to other nucleic acids or to any other type of ligand and they may therefore be suitable as capture agents for use in immunoassays Certain proteins behave as antibody mimetics, therefore have very similar properties but are not antibodies and, as such, the processes herein described may be applicable to such proteins.

Polymers may be considered to be less directly applicable to diagnosis or detection.

However the field of polymer chemistry is so advanced and rapidly developing that it is possible that some polymer will be developed that may compete with any of the other classes of ligands.

Small molecules can be made to be very selective to other small molecules or to any other type of ligand, functional groups or ions which may be applicable to these processes. The chemistries and orientations of small molecules with respect to the surface tend to be more controlled than non-specifically absorbed proteins. This can enable precision-control of the functionality of the particles and subsequent modifications. Small molecules on their own can have affinity to certain markers.

Ions can provide stabilisation to the nanoparticles, while being weakly bound that they can be displaced by another ligand. This can be useful in protocols wherein the ligand bound to the nanoparticle is exchanged for a more stable ligand within an intermediate step.

The ligand may be added in an amount required to form a full monolayer, as part of the process of funcfionalising a nanoparticle. In some embodiments, the ligand may be added in a stoichiometric amount or in slight excess. This can eliminate or reduces nanoparticles conjugates forming aggregates with other nanoparticles conjugates via inter nanoparticle crosslinking. In some embodiments, the ligand can be added in an amount less than what is required to form a full monolayer, especially if multiple ligands are desired in the same layer.

There are a number of ways in which the degree or extent of nanoparticle conjugation is important to the reliable and reproducible functioning of an immunoassay. Firstly the net affinity of the particles depends upon the level of conjugation. In some cases it is desirable that the level of conjugation is maximal, with as many antibodies bound per particle as possible. In other cases however, it may be desirable to tune the dynamic range by having fewer antibodies per nanoparticle, however in order to achieve reproducible results, the level of conjugation needs to be reliably obtained in production of the nanoparticle-antibody conjugate. Similarly it might be desirable to have a nanoparticle capable of working with more than one assay and therefore, for example, a mix of two different antibodies conjugated to the surface could be advantageous. A different type of example comes from the needs of integrated assay systems, where the reagents are placed on or within a disposable cartridge or consumable item. In these cases the reagents are often sequestered from the assay spot either through being in solid form placed in proximity, or placed in solid or liquid or gel form upstream of a flow which carries both sample matrix and reagents to the measurement region of the cartridge or consumable item. The dissolution, flow and adhesion properties of the nanoparticles are therefore also important to the reliable and reproducible functioning of such an integrated assay. In some cases it may be desirable to make the particles more sticky so that they are more easily retained in a location in the device prior to their involvement in the binding of analytes, or in some cases it may be desirable to make them less sticky so that they are more easily carried by flow. A similar situation pertains to dissolution so that the particles can subsequently mix with the analyte matrix by diffusion or other means. In all of these cases, controlled and reproducible levels of conjugation of molecular species which affect, dissolution or non-specific binding is desirable, whilst still allowing controlled levels of conjugation of antibodies too.

In some embodiments, the localised surface plasmon resonance maximum wavelength is between 300 to 1500 nm. The absolute range of wavelengths as well as the AA may be highly dependent on the choice of nanoparticles. For example a single non-interacting spherical gold nanoparticle would have a maximum at around 510 nm to 520 nm, and the AA would be in the region of a few nms. If two spherical gold particles are very close together, the maximum can now be between 520 nm and 800 nm. The closer they are together, the further red-shifted the maximum. Likewise, AA will change strongly depending on their separation, and can be around 100 nm for very close separation. This becomes much more complex to define because particles can be made in a huge variety of other shapes, each of which will have their own maximum and AA.

In some embodiments, the nanoparticle may be a non-spherical, a spherical, a spherical dimer, a nanorod, a prism, or a star-shaped nanoparticle.

Some nanoparticles with a non-spherical shape can exhibit more than one maximum absorbance peak, each of which would have different sensitivities to the refractive index change from ligand binding. For example, a nanorod may exhibit a similar mode to the spherical particle as well as a new mode which will be determined by the size of the long axis. The mode that is also observed in spherical particles (P4520 nm) will have similar sensitivities to the refractive index as will the spherical particles, however the new mode can be more sensitive and have higher AN for the same ligand. Preferably, the nanoparticle is a spherical nanoparticle.

The wavelength of the LSPR maximum absorbance before conjugation may be measured between 0.1 to 200 nm. The wavelength of the LSPR maximum absorbance of a fully conjugated nanoparticle may be measured between 0.1 to 200 nm.

In some embodiments, the nanoparticles are gold nanoparticles. It is advantageous to provide gold nanoparticles due to its unique physical and chemical properties such as its high chemical stability, easy and reproducible preparation and surface modification methods, shape and size controllability, and low-toxicity properties.

In some embodiments, the localised surface plasmon resonance maximum wavelength of a gold nanoparticle, which may be a spherical gold nanoparticle, is between 510 nm to 550 nm.

Alternatively, the nanoparticle can be made of silver. In some embodiments, the localised surface plasmon resonance maximum wavelength of a silver nanoparticle, which may be a spherical silver nanoparticle, is between 390 nm to 450 nm.

Dimers and other higher order aggregates can change the affinity, diffusion coefficients and the particle susceptibility to otherwise insignificant forces, such as gravity. Therefore, monitoring the conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation also requires monitoring of the aggregation. Extra maximum absorbance peaks may arise due to the aggregation. The extent of the aggregation can be obtained through the ratio between single-particle LSPR maximum and an arbitrary red-shifted wavelength from the single particle mode (e.g. 540 nm n and 640 run respectively). The red-shifted wavelength for use in determining the extent of aggregation would be in a region where both the unconjugated and fully conjugated nanoparticles would have a relatively low absorbance and where an aggregated nanoparticle would have an increased absorbance.

Alternatively, the increased absorption due to the aggregates could be fitted and subtracted from the spectrum in order to de-convolute the effect of aggregates and the single particles. The challenge in this arises that each higher order structure will possess a slightly different extinction spectrum. The peak for a d mer could be 700 nm, whereas a linear timer could have a peak at 750 nm, while a triangular trimer could have a peak at 730 nm. The total extinction spectrum that is measured would effectively be a convolution of all these potential bi-products.

However, the processes described herein could be applied to silver or even to copper, titanium nitride, platinum, palladium, gallium and other materials with similar properties.

In another aspect of the invention, there is provided a computer readable medium comprising instructions which, when carried out by a processor, cause the processor to carry out the semi-quantitative process for gauging nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation and the process of functionalising a nanoparticle for use in an immunoassay.

The methods described herein can be performed with a system. For example, a system can include a computer system that comprises: a processor: and a tangible, machine-readable storage medium that stores machine-readable instructions for execution by the processor, the machine-readable instructions corresponding to one or more of the methods described herein. That is, the methods described herein can be performed on computing devices (or processor-based devices) that include a processor; a memory coupled to the processor; and instructions provided to the memory, wherein the instructions are executable by the processor to perform the methods (or steps of the methods) described herein. The instructions can be a portion of code on a non-transitory computer readable medium. Any suitable processor-based device may be utilized for implementing all or a portion of embodiments of the present techniques, including without limitation personal computers, networks personal computers, laptop computers, computer workstations, mobile devices, multi-processor servers or workstations with (or without) shared memory, high performance computers, and the like. Moreover, embodiments may be implemented on application specific integrated circuits (ASICs) or very laroe scale integrated (VLSI) circuits.

The term "computer-readable medium" refers to any tangible storage that participates in providing instructions to a processor for execution. Such a medium may take many forms, including but not limited to, non-volatile media, and volatile media. Non-volatile media includes, for example; NVRArVI, or magnetic or optical disks. Volatile media includes dynamic memory, such as main memory. Computer-readable media may include; for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, magneto-optical medium, a CD-ROM, any other optical medium, a RAM, a PROM, and EPROM, a FLASH-EPROM, a solid state medium like a holographic memory, a memory card, or any other memory chip or cartridge, or any other physical medium from which a computer can read. When the computer-readable medium is configured as a database, it is to be understood that the database may be any type of database, such as relational, hierarchical, object-oriented, and/or the like. Accordingly, exemplary embodiments of the present techniques may be considered to include a tangible storage medium or tangible distribution medium and prior art-recognized equivalents and successor media, in which the software implementations embodying the present techniques are stored.

The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings, in which: Figure 1 shows the random nature of non-specific conjugation, only certain antibodies bound to the nanoparticles will retain their antigen recognition (in this schematic these are highlighted by the dashed antibodies); Figure 2 shows a flow chart describing the semi-quantitative process for gauging nanoparticle conjugation efficiency, the extent of nanoparticle conjugation and/or the degree of nanoparticle conjugation, including the preliminary process steps of calibration; Figure 3 shows a typical absorbance spectrum of spherical gold nanoparticles showing the localised surface plasmon resonance (LSPR) maximum; Figure 4 shows a normalised absorbance spectra between fully conjugated and unconjugated gold nanoparticle-protein sample (dotted and dashed lines, respectively); and Figure 5 shows the ratio of As"/ALspR can be used as a measure of the extent of aggregation.

Referring to Figure 1, there is shown a nanoparticle 16, such as a gold nanoparticle, with the immobilised ligands, such as antibodies 12 and 14 bound to the surface of said nanoparticle 16, wherein some of the antibodies have retained their antigen recognition 12 while other antibodies 14 have not. As shown in Figure 1, the nanoparticle is a spherical nanoparticle 16. The nanoparticle 16 may be any suitable shape and/or size. For example, the nanoparticle can be, but is not limited to, a non-spherical, a spherical, a spherical dimer, a nanorod, a prism, or a star-shaped nanoparticle.

As shown in Figure 1, there is provided a nanoparticle 16. A plurality of ligands are immobilised onto the nanoparticle surface. The ligand immobilised on the substrate have a strong affinity for the ligand bound to the nanoparticles. The ligand can be immobilised onto the surface of the nanoparticle following EDC/NHS activation route, or by non-specific adsorption. Proteins have functionalities within amino acid sequences so that the proteins have very strong binding affinities to the nanoparticles surface. For example, cysteines will chemisorb via the gold-thiol bond. In another example, Lysine will contribute to the strong amine-gold bonding. Other nucleic acids can also contribute to the overall affinity.

Referring to Figure 2, there is shown a flow chart showing the semi-quantitative process for gauging nanoparticle efficiency according to the present invention. The process includes the preliminary steps of calibration, comprising a first step of measuring the wavelength of the localised surface plasmon resonance (LSPR) maximum of bare nanoparticles. As an example only, the measured wavelength may be, but not limited to, between 510 nm and 550 nm. Larger nanoparticles may also have a higher wavelength.

Referring to Figure 2, step (b); If a conjugation efficiency, the extent of conjugation and/or the degree of conjugation scale has already been established for the type of particle that is being conjugated with the desired ligand, then this particle type is not new, and the established scale can be used, and so proceed to step (c). However, if the ligand or the particle is different to any of the conjugation efficiency, the extent of conjugation and/or the degree of conjugation scales that have been recorded before i.e. a different shape, material, initial stabilising agents or composition, then these are considered new, and so, proceed to step (f) as shown in Figure 2.

Referring to Figure 2, step (c); the surface of the nanoparticles is saturated with a ligand such as an antibody. In some examples, the surface of the nanoparticles may be saturated with one or more types of ligand. Step (d) then involves measuring the wavelength of the LSPR maximum of treated nanoparticles and (e) determining the maximum wavelength shift of treatment for the nanoparticle and extent of aggregation.

Alternatively, the step comprising a batch of conjugation of nanoparticles with ligands can be carried out as shown in step (f) in Figure 2. This is then followed by the step of measuring the wavelength of the LSPR maximum of conjugated nanoparticles and determining the extent of aggregation, as shown in step (f). An acceptable level of aggregation can be determined from the calibration standards, namely the unconjugated nanoparticles and the fully conjugated nanoparticles. For the nanoparticle to have an acceptable level of aggregation it should fall between the standards set by the unconjugated nanoparticle and the fully conjugated particle. If it does not, then it would be deemed to not have an acceptable level of aggregation.

The process further comprises the steps of (i) determining the coverage of ligand bound to the nanoparticles through a ratio between the maximum and measured shifts. For example, the target coverage may depend on what is aimed for the conjugation. As an example only, it may be desirable to initially achieve 30% coverage with a ligand that prevents aggregation, followed by a further 50% coverage with a capture agent, such as an antibody. Thus, when the measured coverage is within the acceptable limit, this is an indication that conjugation between ligand and nanoparticle is successful. The steps of the process (f) to (j) as shown in Figure 2 can be repeated until coverage is deemed to be within an acceptable limit.

Partial functionalifies may be required for tuning the overall nanoparticle affinity by decreasing the number of ligands bound to the nanoparticle. Partial funcfionalifies may also be required when carrying out multiplexing of capture agents. Partial functionalities may also be required for reducing or increasing the stickiness of the nanoparticles in the assay through the use of non-sticky or sticky ligands, respectively. Partial functionality can also enable localisation of the nanoparticles in a desired part of a chip, with the rest being capture agents.

Referring to Figure 3, there is shown a typical absorbance spectrum of spherical gold nanoparticles showing the LSPR maximum 18 at 540 nm.

Referring to Figure 4, there is shown a normalised absorbance spectrum, wherein the spectral shift 20 can be calculated from the spectral trace maximum of a fully conjugated gold nanoparticle-protein sample 22 and from the spectral trace maximum of an unconjugated gold nanoparticle-protein sample 24.

Referring to Figure 5, there is shown the absorbance ratio between a single-particle LSPR maximum 26 at 540 nm and an arbitrary red-shifted wavelength from the single particle mode 28 at 640 nm (Aagg/ALspR). This can be used to measure the extent of aggregation.

As an example, the absorbance of a batch of plasmonic nanoparticles in the visible spectrum is measured by a spectrometer (e.g. nanodrop). The same batch of nanoparticles is measured after being incubated in a 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) solution for 1 hour. The value of the localised surface plasmon resonance (LSPR) maximum wavelength is determined by fitting a fourth order polynomial to the spectra between 500 to 600 nm. The extent of aggregation of the BSA protein ligand sample is measured by dividing the absorbance value at the LSPR maximum and the absorbance that is 100 nm red-shifted of this value. The values for the wavelength shift (LA) and the extent of aggregation are taken to be a calibration scale for the batch of nanoparticles. Once the batch of nanoparticles is conjugated with antibodies in either a stoichiometric or a slight excess to what is needed to fully cover the surface, the visible absorbance spectrum is measured in a similar fashion. From the LSPR maximum wavelength of the antibody-conjugated nanoparticles, the coverage is determined by: (Aeon) Abare)

AX

wherein, Aconj is the LSPR maximum wavelength of the conjugated nanoparticles; Abare is the LSPR maximum wavelength of the unconjugated nanoparticles and AX is the wavelength shift of the calibration standard The wavelength shift of the calibration can be calculated by subtracting the wavelength of the LSPR maximum of the unconjugated nanoparticle from the wavelength of the LSPR maximum of the fully conjugated nanoparticle.

The extent of the aggregation is also determined for the conjugated batch in a similar way it was determined for the BSA sample. The batch of conjugated nanoparticles is deemed good enough if the extent of aggregation of the conjugated is also not changed by more than 20 % than that of the calibration standard.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

CLAIMS1. A semi-quantitative process for gauging nanoparticle conjugation extent, degree or efficiency comprising the steps of: i. measuring a spectral shift of the localised surface plasmon resonance (LSPR) maximum absorbance of an unconjugated nanoparticle and a fully conjugated nanoparticle; and converting the spectral shift into a value of conjugation efficiency by comparison with a predetermined scale of nanoparticle coverage.
2. The process according to claim 1, further comprising the preliminary steps of: i. measuring the wavelength of the maximum absorbance of the LSPR in a spectrum before conjugation; ii. measuring the wavelength of the maximum absorbance of the LSPR in a spectrum of a fully conjugated nanoparticle; and iii. determining the conjugation scale of nanoparticle coverage.
3. The process according to claim 2, wherein the step of determining the conjugation scale of nanoparticle coverage comprises determining the difference of the spectrum before conjugation and of a fully conjugated nanoparticle.
4. A process of functionalising a nanoparticle for use in an immunoassay, the process comprising the steps of: i. applying a first layer of ligands; and ii. gauging the nanoparticle conjugation efficiency using the process of any one of claims 1 to 3.
5. The process according to claim 4, further comprising the steps of: i. applying a further layer of ligands; and ii. gauging the nanoparticle conjugation efficiency of the further layer of ligands using the process of any one of claims 1 to 3.
6. The process according to claim 4 or claim 5, wherein the ligands in each layer are all the 5 same.
7. The process according to any preceding claim, wherein the ligand is an antibody.
8. The process according to any preceding claim, wherein the ligand is a nucleic acid, protein, polymer, small molecule or ion.
9. The process according to any preceding claim, wherein the localised surface plasmon resonance maximum wavelength is between 300 to 1500 nm.
10. The process according to any one of the preceding claims, wherein the nanoparticle is a non-spherical, a spherical, a spherical dimer, a nanorod, a prism, or a star-shaped nanoparticle.
11. The process according to claim 10, wherein the nanoparticle is a spherical nanoparticle.
12. The process according to any one of the preceding claims, wherein the nanoparticle is made of gold.
13. The process according to claim 12, wherein the localised surface plasmon resonance maximum wavelength of a gold nanoparticle is between 510 nm to 550 nm.
14. The method according to claims 1 to 11, wherein the nanoparticle is made of silver.
15. The method according to claim 14, wherein the localised surface plasmon resonance maximum wavelength of a silver nanoparticle is between 390 nm to 450 nm.
16. The process according to any preceding claim, wherein the wavelength of the LSPR maximum absorbance before conjugation is measured between 0.1 to 200 nm.
17. The process according to any preceding claim, wherein the wavelength of the LSPR maximum absorbance of a fully conjugated nanoparticle is measured between 0.1 to 200 10 nm.
18. A computer readable medium comprising instructions which, when carried out by a processor, cause the processor to carry out the processes according to any one of the preceding claims.