Surface generation

Abstract

A method of generating a binding surface for an object in a solution comprising the steps of: i. obtaining a blocked surface wherein the blocked surface comprises a base surface and at least one blocking reagent; ii. targeting of a portion of the blocked surface with a light source; and iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the binding rate of the object to the blocked surface is less than the binding rate of the object to the base surface is provided. A method of generating a binding surface for an object in a solution is also provided.

Description

[0001] SURFACE GENERATION

[0002] Field of the invention

[0003] The present invention relates to methods and products for mass photometry, in particular for taking multiple mass photometry measurements on the same base surface, e.g. in a microfluidic channel.

[0004] Background to the invention

[0005] Mass photometry is a useful tool for assessing the mass of objects in solution, such as proteins, nucleic acids and nanoparticles. The technique comprises exposing an object in solution to a surface and detecting the binding and unbinding of the object to the surface using interferometric scanning microscopy (iSCAT) (W02018 / 011591 , WO2019 / 110977, Young et al., Science (April 2018) 360(6387), 423-427 and Li et al., Nucleic Acids Research (August 2020) 48(17), e97). One drawback of Mass photometry is that some objects adhere strongly to the surface and can accumulate over time. The surface can become saturated and thus unusable for further experiments and samples can also become contaminated with previously measured objects. The surface must therefore be regularly cleaned to remove bound objects. However, the cleaning process may involve substances or reagents that can damage the surface which would impact the next iSCAT measurement.

[0006] It is against this background that the present invention has arisen.

[0007] The present invention resolves these issues by creating a blocking layer covering the surface, a portion of which can be selectively removed using a laser to create a “single-use” binding area. This can be repeated several times across the surface allowing for a sequence of measurements to be taken from a single surface wherein each measurement is made on a “virgin” binding surface. of the invention

[0008] The present invention relates to methods and products for measuring an object when adhered to a surface using any techniques, such as mass photometry. In particular, there is provided a method for generating a binding surface by removing a blocking reagent from a base surface to allow multiple mass photometry measurements on the same base surface, without the need for cleaning of the base surface.

[0009] In an aspect of the present invention, there is provided a method of generating a binding surface for an object in a solution comprising the steps of: i. obtaining a blocked surface wherein the blocked surface comprises a base surface and at least one blocking reagent; ii. targeting of a portion of the blocked surface with a light source; and iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the rate of binding of the object to the blocked surface is less than the binding rate of the object to the base surface.

[0010] In another aspect of the present invention, there is provided a method of generating a binding surface for an object in a solution comprising the steps of: i. obtaining a blocked surface wherein the blocked surface comprises a base surface and at least one blocking reagent; ii. targeting of a portion of the blocked surface with a light source; and iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the rate of medium-lived and / or long-lived binding of the object to the blocked surface is less than the binding rate of the object to the base surface.

[0011] As referred to herein, and unless otherwise specified, the “blocked” surface of the present invention can act to prevent permanent interactions, binding or attachment of an object to the blocked surface or region and / or to prevent an accumulation of one or more objects on the blocked surface. In some instances, the blocked surface may have transient or temporary interactions with the object but the rate of binding is substantially equal to the rate of unbinding and thus, the object is not permanently bound to the blocked surface for any substantial period of time. Therefore, the rate of binding of an object to the blocked surface will significantly be less than the binding rate of the object to the non-blocked i.e. base surface. In some instances, the rate of medium-lived to long-lived binding event of an object to the blocked surface shall be zero or near-zero.

[0012] In some embodiments the binding rate of the object to the blocked surface is less than 20% of the binding rate of the object to the base surface. Suitably the binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface. Suitably the binding rate of the object to the blocked surface is less than 5% of the binding rate of the object to the base surface. Suitably the binding rate of the object to the blocked surface is less than 2% of the binding rate of the object to the base surface. Suitably the binding rate of the object to the blocked surface is less than 1% of the binding rate of the object to the base surface. In some embodiments, the binding rate of the object to the blocked surface is at least 0.1% and less than 20% of the binding rate of the object to the base surface.

[0013] In some embodiments the net binding rate of the object to the blocked surface is less than 20% of the binding rate of the object to the base surface. Suitably the net binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface. Suitably the net binding rate of the object to the blocked surface is less than 5% of the binding rate of the object to the base surface. Suitably the net binding rate of the object to the blocked surface is less than 2% of the binding rate of the object to the base surface. Suitably the binding rate of the object to the blocked surface is less than 1% of the binding rate of the object to the base surface. In some embodiments, the net binding rate of the object to the blocked surface is at least 0.1 % and less than 20% of the binding rate of the object to the base surface. Where net binding events is the sum of unbinding events subtracted from the sum of binding events and refers to the overall accumulation of the object on the surface.

[0014] In some embodiments the base surface comprises a glass surface which may be the surface of a glass slide, the surface of a solid immersion lens, or the surface of a flow cell. Such surfaces are suitable for obtaining mass photometry measurements.

[0015] In some embodiments where the base surface comprises a glass surface the base surface further comprises a layer of an intermediary reagent configured to form an interface between the glass surface and the blocking reagent, optionally wherein the layer of intermediary reagent is not substantially removed by step iii.

[0016] In some embodiments, the blocked surface may be prepared by functionalising the base surface with an intermediary reagent layer and then functionalising the intermediary reagent layer with a blocking reagent layer. Alternatively, the blocked surface may be prepared by first functionalising the intermediary reagent with the blocking reagent and then functionalising the base surface with the combined intermediary and blocking reagent layer.

[0017] The intermediary reagent may comprise an amine moiety. The intermediary may comprise a silane moiety. The intermediary reagent may comprise both amine and silane moieties. The intermediary reagent may be a compound according to Formula A

[0018] Formula A

[0019] Wherein n is 2-6 and R1, R2, and R3are independently selected from Ci-Ce alkyl or alkoxy. The Ci-Ce alkyl or alkoxy may be branched or may be unbranched. Optionally R1, R2, and R3are all methoxy. Optionally R1, R2, and R3are all ethoxy. Optionally R1is a Ci-Ce alkoxy group and R2and R3are Ci-Ce alkyl groups. Optionally n is 3.

[0020] The term "alkyl" as used herein and unless otherwise specified, means straight or branched chain, saturated alkyl groups. The term "alkoxy" as used herein and unless otherwise specified, means straight or branched chain, saturated alkoxy groups.

[0021] In some embodiments, the layer of intermediary reagent may comprise a 3- aminopropyltrimethoxysilane (APTMS). Alternatively, the layer of intermediary reagent may comprise a 3-aminopropyldiisopropylethoxysilane (APDIES) layer. Alternatively, the layer of intermediary reagent may comprise a 3-aminopropyldimethylethoxysilane (APDMES) layer.

[0022] An intermediary reagent increases the diversity of blocking reagents which are compatible with the present invention beyond those which natively adhere to the glass surface.

[0023] In some embodiments the blocking reagent is selected from bovine serum albumin (BSA), polyethylene-glycol (PEG), or polyoxazoline. The polyoxazoline may be polymethyloxazoline (PMOXA). Such blocking reagents are inexpensive and widely available and form effective blocking layers. In such embodiments, during step iii, the space immediately above the portion of the blocked surface may comprise air. In some embodiments, the space above the portion of the blocked surface may comprise air shortly after step iii. This enables more effective removal of the blocking reagent from the base surface.

[0024] In some embodiments the blocking reagent comprises a photolabile reagent. Suitably the photolabile reagent is a photolabile polyethylene glycol. The photolabile polyethylene glycol may be a compound of Formula B:

[0025] Formula B wherein R is a terminal group and n is 1-50.

[0026] In the compounds of Formula B, R can be selected from the group consisting of: azide (N3), methoxy, thiol or tert-butoxycarbonyl.

[0027] In the compounds of Formula B, n is 1-50, for example, 1-40, 1-30, 5-50, 5-40, 5-30, 10-50, 10-40 or 10-30.

[0028] Suitably, the photolabile polyethylene glycol may be a PC (photocleavable) azidopentafluorophenyl (PFP) ester. Suitably the photolabile polyethylene glycol may be a PC (photocleavable) azido-PEG-NHS carbonate ester:

[0029] Formula C

[0030] Suitably the PC azido-PEG-NHS carbonate ester is PC Azido-PEG11-NHS carbonate ester (n=11) or PC Azido-PEG23-NHS carbonate ester (n=23).

[0031] In some embodiments, PEG may be functionalised using standard solution phase methods. Alternatively, PEG may be functionalised at cloud point. Functionalising at the cloud point for PEG decreases the hydrodynamic radius of PEG in solution, thereby allowing the PEG chains to pack closer together and increase the PEG brush density on a surface. In some embodiments, PEG functionalisation at cloud point may therefore lead to an improved blocking performance of the functionalised PEG reagent.

[0032] Suitably the photolabile reagent may be a photolabile polyoxazoline. The photolabile polyoxazoline may be a photocleavable polymethyloxazoline-activated ester. The photolabile polyoxazoline may be a photocleavable polymethyloxazoline-NHS carbonate ester (PC- PMOXA-NHS carbonate ester) or a photocleavable polymethyloxazoline-pentafluorophenyl (PC-PMOXA-PFP ester), or a photocleavable polymethyloxazoline-tetrafluorophenyl (PC- PMOXA-TFP ester), or a photocleavable polymethyloxazoline-N-hydroxysuccinimide- succinimidyl valerate (PC-PMOXA-NHS-SVA) ester.

[0033] For example, the photolabile reagent may be photocleavable polymethyloxazoline-NHS carbonate ester (PC-PMOXA-NHS carbonate ester) as shown in Formula D:

[0034] Formula D

[0035] Wherein n is 1-50 for example, 1-40, 1-30, 5-50, 5-40, 5-30, 10-50, 10-40 or 10-30.

[0036] Suitably n is 50 and the photocleavable polyoxazoline-NHS carbonate ester may be photocleavable polymethyloxazoline-NHS carbonate ester (PC-PMOXA50-NHS carbonate ester) as shown in Formula E:

[0037] Formula E

[0038] In some embodiments, the photolabile group may include, but is not limited to, nitrobenzyl groups such as o-Nitroveratryloxycarbonyl (NVOC), 2-(2-Nitrophenyl)- propyloxycarbonyl (NPPOC), Benzoin, (coumarin-4-yl)methyl, 7-nitroindoline or p- hydroxyphenacyl.

[0039] In such embodiments, during step iii, the space immediately above the portion of the blocked surface may comprise a liquid medium.

[0040] Photolabile blocking reagents can be removed from the base surface more readily than other blocking reagents due to their inherent properties. They can be removed from the base surface whilst a liquid medium is present.

[0041] In some embodiments, the blocked surface may comprise a further blocking reagent or a binding modifier. For example, the blocked surface may comprise both a photolabile blocking reagent and a non-photolabile blocking reagent. In some embodiments, the non-photolabile blocking reagent may remain at the base surface after the photolabile blocking agent has been removed. The blocked surface may comprise a photolabile blocking reagent and a binding modifier, wherein the binding modifier is not removed during step iii and enhances binding of particular objects once the photolabile blocking agent is removed.

[0042] In an embodiment the blocked surface comprises a photolabile polyethylene glycol and a non- photolabile acetate such as sulpho-NHS-acetate.

[0043] In some embodiments, the blocked surface may comprise a plurality of different photolabile blocking reagents. For example, the blocked surface may comprise both photolabile polyethylene glycol and a photolabile non-volatile organic compound (NVOC). The photolabile NVOC may be a photolabile acetate, azide, alkyl, or other suitable group. A suitable photolabile acetate may be sulfo-NHS-NVOC-acetate or may be a non-sulfo-NHS-NVOC-acetate. A suitable photolabile azide may be N-(5-azido-2-nitro-benzoyloxy)succinimide. A suitable photolabile alkyl may be 4,5-Dimethoxy-2-nitrobenzyl chloroform ate. Due to their steric bulk, large blocking reagents such as polyethylene glycols may not saturate the blocked surface or react with all reactive groups of the intermediary layer. The addition of a further blocking reagent such as a relatively small photolabile NVOC enables unreacted intermediary reagent to be quenched. A blocked layer comprising a plurality of different photolabile blocking reagents may improve the blocking for mRNA and / or DNA macromolecules.

[0044] In some embodiments step iii comprises irradiating the portion of the blocked surface with visible light or with near-UV light. The near-UV light may have a wavelength of 315 to 400 nm. Suitably the near-UV light may have a wavelength of 375 nm. The visible light may have a wavelength of 450 to 550 nm. Suitably the visible light may have a wavelength of 470 to 500 nm. Suitably the visible light may have a wavelength of 480 to 490 nm. Suitably the visible light may have a wavelength of 488 nm. Such wavelengths of light are readily generated and are suitable for effective removal of the blocking reagent.

[0045] In some embodiments step iii comprises irradiating the portion of the blocked surface with light of a power density of greater than 1 KWITT2. When the blocked surface comprises a photolabile reagent suitably the power density is 1 KWm’2to 5 GWITT2.

[0046] When the blocked surface comprises a photolabile reagent and near UV light is used in step iii, the power density is suitably 1 KWm-2to 2 MWm-2. Suitably the power density is 10 KWrrr2to 1 MWOT2Suitably the power density is about 500 KWm-2. Such power densities are suitable for effective and rapid removal of blocking reagents such as PC-PMOXA-NHS carbonate ester.

[0047] When the blocked surface comprises a photolabile reagent and visible light is used in step iii, suitably the power density is 1 to 5 GWm-2. Suitably the power density is 2 to 4 GWOT2. Suitably the power density is about 2.5 GWm-2. Such power densities are suitable for effective and rapid removal of most photolabile blocking reagents.

[0048] When the blocking reagent does not comprise a photolabile reagent and visible light is used during step iii, suitably the power density is 1 to 12.5 GWm’2. Suitably the power density is 2 to 10 GWm’2. Such power densities are suitable for effective and rapid removal of most blocking reagents including non-photolabile blocking reagents.

[0049] In some embodiments step iii comprises irradiating the portion of the blocked surface for at least 0.1 second. In some embodiments, step iii comprises irradiating the portion of the blocked surface for less than 10 seconds. In some embodiments, step iii comprises irradiating the portion of the blocked surface for less than 120 seconds. When the blocking reagent comprises a photolabile reagent suitably the portion of the blocked surface is irradiated for 0.1 to 30 seconds. Suitably the portion of the blocked surface is irradiated for 1 to 20 seconds. Suitably the portion of the blocked surface is irradiated for 5 to 10 seconds. When the blocking reagent does not comprise a photolabile reagent suitably the portion of the blocked surface is irradiated for 30 to 180 seconds. Suitably the portion of the blocked surface is irradiated for 60 to 120 seconds. Such durations are suitable for effective removal of a portion of the blocking reagent and are short enough to allow high throughput sample processing.

[0050] In some embodiments step iii comprises irradiating the portion of the blocked surface with at least 200 KJm-2energy. When the blocking reagent comprises a photolabile reagent, suitably the portion of the blocked surface is irradiated with 200 KJm-2to 150 GJnr2energy.

[0051] When the blocked surface comprises a photolabile reagent and near UV light is used in step iii, the blocked surface is suitably irradiated with 200 KJm-2to 250 MJnr2energy. Suitably the blocked surface is irradiated with 1 to 100 MJnr2energy. Suitably the blocked surface is irradiated with about 10 MJnr2energy. Such energies are suitable for effective of blocking reagents such as PC-PMOXA-NHS carbonate ester.

[0052] When the blocked surface comprises a photolabile reagent and visible light is used in step iii, suitably the portion of the blocked surface is irradiated with 1 to 150 GJm-2energy. Suitably the blocked surface is irradiated with 5 to 100 GJm-2energy. Suitably the portion of the blocked surface is irradiated with 10 to 50 gJm-2energy. Suitably the blocked surface is irradiated with about 25 GJm-2energy. Such energies are suitable for effective of most photolabile blocking reagents.

[0053] When the blocking reagent does not comprise a photolabile reagent and visible light is used during step iii, suitably the portion of the blocked surface is irradiated with 60 to 4500 GJnr2energy. Suitably the portion of the blocked surface is irradiated with 500 to 3000 GJnr2energy. Suitably the portion of the blocked surface is irradiated with about 1500 GJnr2energy. Such energies are suitable for effective of most blocking reagents including non-photolabile blocking reagents.

[0054] In some embodiments the object is a macromolecule. The macromolecule may be a protein or a complex of proteins. Such objects are generally of a size which can be detected using mass photometry and are interesting targets for study. The macromolecule may comprise nucleic acids including DNA or RNA. The macromolecule may be mRNA.

[0055] The invention also provides a method of performing a mass photometry measurement of an object in solution comprising the method of generating a binding surface disclosed above and: iv. adding the object in solution to the binding surface; and v. making a mass photometry measurement.

[0056] This method means that a fresh binding surface is generated for the object in solution to bind to and be measured by mass photometry, thus minimising sample contamination.

[0057] In some embodiments the steps of the method of performing a mass photometry measurement of an object in solution proceed from i. to v.

[0058] In other embodiments the steps of the method of performing a mass photometry measurement of an object in solution are carried out in a different order, for example step iv. may precede step iii. or step ii. meaning the object in solution is present on the blocked surface before the unblocking step occurs. This allows the blocking reagent to be removed at a pre-determined time and thus for a time-resolved mass photometry measurement of the object in solution to be obtained. In such embodiments the blocking reagent suitably comprises a photolabile moiety which allows for facile unblocking of the blocked surface and thus greater time resolution of the mass photometry measurement and allows for unblocking of the blocked surface when a liquid medium is present (i.e. the solution of the object in solution).

[0059] The invention also provides a method of performing n mass photometry measurements of an object or objects in solution comprising the method of performing a mass photometry measurement of an object in solution disclosed above followed by n-1 cycles of steps ii-iv; wherein each iteration of step ii targets a portion of the blocked surface which is spatially distinct from any previously targeted portions of the blocked surface; and wherein the object in solution in each iteration of step iv is independently selected from a set of m objects where m < n. This method means that a fresh binding surface is generated for each object in solution to bind to even when making multiple measurements using a single base surface.

[0060] In some embodiments the steps of method of performing n mass photometry measurements of an object or objects in solution proceed from i. to v. for the first measurement and ii. to v. for the subsequent iterations.

[0061] In other embodiments, for at least one iteration, the steps of method of an object or objects in solution are carried out in a different order, for example step iv. may precede step iii. or step ii. meaning the object in solution is present on the blocked surface before the unblocking step occurs. This allows the blocking reagent to be removed at a pre-determined time and thus for a time-resolved mass photometry measurement of the object in solution to be obtained.

[0062] In some embodiments, for at least one iteration, step iv. is not carried out and a further mass photometry measurement of the object in solution from the previous iteration is obtained. This further mass photometry measurement may be obtained at a pre-determined time and thus allows for a time-resolved mass photometry measurement of the object in solution to be obtained. In such embodiments the blocking reagent suitably comprises a photolabile moiety which allows for facile unblocking of the blocked surface and thus greater time resolution of the mass photometry measurement and allows for unblocking of the blocked surface when a liquid medium is present (i.e. the solution of the object in solution). n is theoretically limited only by the geometry of the blocked surface and the size of the portions of the blocked surface which are irradiated. In some embodiments n is 384 or less. In some embodiments n is 96 or less. In some embodiments n is 48 or less. These numbers increase compatibility with standard sample processing techniques which make use of plates with 384, 96 48, or fewer wells.

[0063] In some embodiments the method is carried out within a flow cell having a flow defining an upstream direction and a downstream direction. In such embodiments, the portion of the blocked surface targeted in an iteration p+1 of step ii may be in the upstream direction from the portion of the blocked surface targeted in iteration p of step ii; and / or the portion of the blocked surface targeted in an iteration q+1 of step ii may be offset from the portion of the blocked surface targeted in iteration q of step ii in a direction perpendicular to the flow. In these ways, the binding surface generated from the portion of the blocked surface which is targeted in iteration p or q of step ii is not able to be contaminated by any object or reagent which may be released from the binding surface which is generated in iteration p-1 or q-1 because of the effect of the flow.

[0064] The invention also provides a glass surface for binding to an object in a solution, wherein the glass surface comprises: i. a blocked surface comprising a base surface and at least one blocking reagent; and optionally ii. a binding surface wherein a portion of the blocked surface has been irradiated with a light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the binding rate or the net binding rate of the object to the blocked surface is less than the binding rate of the object to the base surface.

[0065] In some embodiments the binding rate or net binding rate of the object to the blocked surface is less than 20% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 5% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 2% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 1 % of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is at least 0.1% and less than 20% of the binding rate of the object to the base surface.

[0066] In some embodiments, the glass surface is the surface of a glass slide, the surface of a solid immersion lens, or the surface of a flow cell.

[0067] The invention also provides a flow cell comprising a glass surface as described above.

[0068] The invention also provides a method of generating a binding surface for an object in a solution comprising the steps of: i. adding a blocking reagent to a base surface to form a blocked surface; ii. targeting of a portion of the blocked surface with a light source; iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the binding rate of the object to the blocked surface is less than the binding rate of the object to the base surface.

[0069] In some embodiments the binding rate or net binding rate of the object to the blocked surface is less than 20% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 5% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 2% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 1 % of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is at least 0.1% and less than 10% of the binding rate of the object to the base surface.

[0070] Brief description of the Figures

[0071] The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings, in which:

[0072] Figure 1 shows a flow chart of the unblocking process;

[0073] Figure 2a-d shows a flow chart of the unblocking process represent the use of flow direction prevent to cross-interaction free mass photometry measurements in this invention;

[0074] Figures 3a and 3b show the effect of sample buildup under standard mass photometry conditions when reusing a surface;

[0075] Figure 4 shows the detected binding events from 96 mass photometry measurements of Bovine IgG using a method of the claimed invention with PEG as the blocking reagent;

[0076] Figure 5 shows the detected binding events from 96 mass photometry measurements of thyroglobulin using a method of the invention with BSA as the blocking reagent; Figure 6 shows the detected binding events from 96 mass photometry measurements of Bovine IgG using a method of the invention with PC Azido-PEG11-NHS carbonate ester as the blocking reagent;

[0077] Figures 7a and 7b show the time dependence of removal of [a] PC Azido-PEG11-NHS carbonate ester in the presence of PBS and [b] PEG (5000 Da) in the presence of air by detection of binding events from mass photometry measurements of bovine IgG;

[0078] Figure 8 shows the detected binding events from 167 mass photometry measurements of RNA using a method of the invention with PC-PMOXA50-NHS carbonate ester as the blocking reagent; and

[0079] Figure 9 shows mass photometry histograms for measurements using the method of the invention and Bovine IgG with PC Azido-PEG23-NHS carbonate ester blocking reagent which was functionalised at cloud point.

[0080] Detailed description of the invention

[0081] The invention provides a method of generating a binding surface for an object in a solution comprising the steps of: i. obtaining a blocked surface wherein the blocked surface comprises a base surface and at least one blocking reagent; ii. targeting of a portion of the blocked surface with a light source; and iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the binding rate of the object to the blocked surface is less than the binding rate of the object to the base surface.

[0082] The steps of the method are shown in the “Unblock Surface” section of Figure 1.

[0083] The base surface may be any surface which is suitable for making mass photometry measurements, to which a blocking reagent can be applied to create a blocked surface, and subsequently to which light can be applied to remove a quantity of the blocking reagent to create a binding surface. The base surface may comprise a glass surface which may optionally be selected from the surface of a glass slide, the surface of a solid immersion lens or the surface of a flow cell.

[0084] The binding surface is a surface which objects can bind to as distinct from a blocked surface which comprises blocking reagents that hinder the interaction of objects with the surface.

[0085] Object, as used herein, means any object which has a mass that can be reliably detected and quantified by mass photometry. The first object should therefore have a mass greater than 10 kDa. The first object may be selected from a macromolecule, a polypeptide, a protein or a fragment thereof, a lipoprotein, a glycoprotein, a complex of proteins, an antibody or an antibody fragment thereof, an enzyme, a nucleic acid, a protein-nucleic acid complex, a virus or portion thereof, a virus-like particle, a viral vector such, an exosome, a cell, a nanoparticle, a quantum dot, a liposome, a vesicle, a micelle, a lipid, a carbohydrate, a polysaccharide, an organic polymer, a compound or an ion.

[0086] The object is in solution as this is necessary for mass photometry measurements. For larger objects such as cells and nanoparticles which are above the size limit to be able to truly dissolve, “in solution” may be substituted with “in suspension”.

[0087] A blocked surface is a surface which comprises a layer of blocking reagent which acts to prevent permanent attachment or binding of objects t the surface, to such an extent that the binding rate or net binding rate of objects to the blocked surface is less than the binding rate of objects to the underlying surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 20% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 5% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 2% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 1% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is at least 0.1 % and less than 20% of the binding rate of the object to the base surface. The binding rate or net binding rate of an object to the surface can be measured using mass photometry and / or any other suitable techniques. The blocked surface may comprise a layer of intermediary reagent configured to form an interface between the base surface and the blocking reagent. Depending on the physical / chemical properties of the base surface and the blocking reagent a secure layer of blocking reagent may not be able to form. Thus, a layer of an intermediary reagent which binds to both the base surface and the blocking reagent may be used. The intermediary reagent can be any reagent which performs this function. For example, the intermediary reagent may comprise both an amine and a silane moiety. The intermediary reagent may be a compound according to Formula A as defined above. Optionally, the layer of intermediary reagent is not entirely or substantially removed by step iii and thus the binding surface may comprise some amount of intermediary reagent. In some embodiments, therefore, the intermediary reagent does not impede the binding of objects to the binding surface. In some embodiments the intermediary layer may enhance binding of the object to the binding surface, for example when the intermediary reagent comprises an amine group this may enhance the binding of DNA or other nucleic acids to the binding surface.

[0088] The blocking reagent may be any reagent which can form a layer on a base surface, or a layer of intermediary reagent if necessary, and thereby impede the binding of objects to the base surface. The blocking layer must also be capable of removal from the blocked surface using light. The blocking reagent may be a protein or polypeptide, a polymer or oligomer, a perfluorinated molecule, a zwitteronic, anionic or cationic molecule, a hydroxyl, a self assembled monolayer (SAM) or a lipid bilayer (e.g. supported lipid bilayer, SLB). The blocking reagent may be BSA or PEG. The blocking reagent may comprise a moiety which enhances binding to the base surface or layer of intermediary reagent. The blocking reagent may comprise a photolabile reagent meaning it contains a chemical moiety that breaks upon exposure to a particular wavelength of light. Suitably the photolabile reagent is a photolabile polyethylene-glycol. Suitably the photolabile polyethylene-glycol is a PC azido-PEG. Suitably the PC azido-PEG has an NHS-carbonate linker. Suitably the PC azido-PEG is PC azido- PEG-NHS carbonate ester. Suitably the photolabile reagent is a photolabile polyoxazoline. The photolabile polyoxazoline may be photocleavable polymethyloxazoline-NHS carbonate ester (PC-PMOXA-NHS carbonate ester).

[0089] Obtaining a blocked surface may comprise preparing a blocked surface. Preparing a blocked surface may include steps such as: generating a layer of intermediary reagent on a base surface by: adding to a base surface a solution comprising at least one intermediary reagent and either soaking for a sufficient amount of time such that a layer of the intermediary reagent forms on the base surface; flowing the solution of intermediary reagent over the base surface for a sufficient amount of time at a sufficient flow rate such that a layer of the intermediary reagent forms on the base surface; depositing a layer of intermediary reagent on a base surface by chemical vapour deposition; adding to a base surface, optionally comprising a layer of intermediary reagent, a solution comprising at least one blocking reagent and either contacting for a sufficient amount of time or flowing the solution of blocking reagent over the base surface for a sufficient amount of time at a sufficient flow rate such that a layer of the blocking reagent forms on the base surface; adding to any surface comprising a layer of blocking reagent a solution comprising at least one secondary blocking reagent and either contacting for a sufficient amount of time or flowing the solution of secondary blocking reagent over the surface for a sufficient amount of time at a sufficient flow rate such that the secondary blocking reagent fills gaps in the layer of blocking reagent; or any other appropriate method.

[0090] Obtaining a blocked surface may alternatively comprise procuring a blocked surface from a supplier.

[0091] Targeting a portion of the blocked surface means selecting a portion of the blocked surface and aiming a light source at said portion. The portion may be any shape or size however a quadrilateral, portion is preferred, and a rectangular portion is most preferred. Suitably the portion of the blocked surface is of the same or similar size to the field of view of a mass photometry device. The light source must be accurately targetable to the portion of the blocked surface which may be achieved with precision optics techniques. The light source is suitably a laser. The light source is suitably a monochromatic laser Suitably the light source is a diode laser. The light source must be capable of generating sufficient power density to remove a quantity of the blocking reagent from the portion of the blocked surface.

[0092] Irradiating the portion of the blocked surface means that the portion of the blocked surface is exposed to a particular power density of light for a particular length of time. Power density means the energy per second being shone on the portion of the blocked surface per unit area. This is preferably measured in W / r2. Suitably the light is monochromatic and of a known wavelength. The combination of the power density, duration and wavelength of the irradiation must be sufficient to remove a quantity of the blocking reagent bound to the portion of the blocked surface. Each of these parameters which vary depending on, at least, the identity of the blocking reagent, the nature of the base surface, and how much blocking reagent is required to be removed. The identity of the blocking reagent in particular has a large effect on the parameters and other conditions which should be used. Non-photolabile blocking reagents for example tend to require greater power densities and / or greater durations of irradiation than photolabile blocking reagents. We have also determined that non-photolabile blocking reagents are best removed from the portion of the blocked surface whilst the space immediately above the portion of the blocked surface comprises air. In some embodiments, the space above the portion of the blocked surface may comprise air during or shortly after irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed.

[0093] On the other hand, photolabile blocking reagents can be removed from the portion of the blocked surface whilst the space immediately above the portion of the blocked surface comprises a liquid medium.

[0094] Without being bound by theory, this is presumably because the non-photolabile reagents are removed because the high energy densities produce heat during irradiation of the portion of the blocked surface which results in the degradation and / or detachment of the blocking reagent. In the presence of a covering solution this heat is absorbed. Photolabile blocking reagents can be removed using lower amounts of irradiation even in the presence of a covering solution because of their light-specific moieties.

[0095] The quantity of blocking reagent removed from the portion of the blocked surface may be all, substantially all, or a fraction of the total amount of blocking reagent. Within the context of the present invention, the term “cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed" should be understood to mean that at least one complete molecule of blocking reagent is detached, displaced or eliminated. In other words, step iii causes at least one molecule of blocking reagent to no longer be present on the blocked surface in its original position. It should be understood that this is distinct from partial disruption of a blocking reagent molecule, or photocleaving of a part of the blocking reagent molecule which do not cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed. In embodiments in which a layer of intermediary reagent is present, causing a quantity of the blocking reagent bound to the portion of the blocked surface to be removed may be understood to mean that a quantity of the blocking reagent is removed to reveal the intermediary reagent layer. The quantity of blocking reagent which is removed from the portion of the blocked surface may be varied by adjusting the parameters of the irradiation. For example, decreasing the power density or duration or irradiation in order to reduce the amount of blocking reagent which is removed. This may be preferable if, for example, a mass photometry measurement is desired of a solution comprising a high concentration of an object, or an object with a particular affinity for the binding surface. In this scenario complete removal of the blocking reagent could lead to overloading of the binding surface and counts which are too high for accurate mass photometry measurements. Incomplete removal of the blocking reagent would allow some binding of the object to the binding surface for measurement whilst keeping the counts within the range of the mass photometry instrument.

[0096] The blocking reagent which is removed from the portion of the blocked surface may be removed uniformly across the portion of the blocked surface or may be weighted towards a sub-region of the portion of the blocked surface. For example, a small amount of blocking reagent may be removed from one side of the portion of the blocked surface whilst total, or near total removal, of the blocking reagent may be performed on the other side of the portion of the blocked surface. There may be intermediate regions or a gradient of blocking reagent removal from one side of the portion of the blocked are to the other. The variation in blocking reagent removal may take any shape, for example it may be a radial variation rather than a linear variation. This variation may be achieved by irradiating different sub-regions of the portion of the blocked surfaces with different wavelengths, with different power densities or for different durations. In this way the binding surface comprises at least two sub-regions with different binding rates for the object. Such a binding surface may be useful for mass photometry measurements of samples where the concentration of the object to be measured is unknown. Having a variable binding rate across the binding surface increases the likelihood of collecting data with a suitable number of counts from at least one sub-region of the binding surface.

[0097] In some embodiments, the blocked surface may comprise a further blocking reagent or a binding modifier. For example, the blocked surface may comprise both a photolabile blocking reagent and a non-photolabile blocking reagents. In some embodiments, the non-photolabile blocking reagent may remain at the base surface after the photolabile blocking agent has been removed. The blocked surface may comprise a photolabile blocking reagent and a binding modifier, wherein the binding modifier is not removed during step iii and enhances binding of particular objects once the photolabile blocking agent is removed. In an embodiment the blocked surface comprises a photolabile polyethylene glycol and a binding modifier. The binding modifier may comprise terminal alkyl groups. In some embodiments, the non- photolabile blocking reagent or binding modifier may improve binding for proteins.

[0098] In some embodiments, the blocked surface may comprise a plurality of different photolabile blocking reagents. For example, the blocked surface may comprise both photolabile polyethylene glycol and a photolabile non-volatile organic compound (NVOC). The photolabile NVOC may be a photolabile acetate, azide, alkyl or other suitable group. A suitable photolabile acetate may be sulfo-NHS-NVOC-acetate or non-sulfo-NHS-NVOC acetate. A suitable photolabile azide may be N-(5-azido-2-nitro-benzoyloxy)succinimide. A suitable photolabile alkyl may be 4,5-Dimethoxy-2-nitrobenzyl chloroform ate. Due to their steric bulk, large blocking reagents such as polyethylene glycols may not saturate the blocked surface or react with all reactive groups of the intermediary layer. The addition of a further blocking reagent such as a relatively small photolabile NVOC enables unreacted intermediary reagent to be quenched. A blocked layer comprising a plurality of different photolabile blocking reagents may improve the blocking for mRNA and / or DNA macromolecules.

[0099] Suitably the light used for the irradiation of the portion of the blocked surface is in the visible or near-UV regions of the electromagnetic spectrum, i.e. between 200 and 800 nm. The near- UV light may have a wavelength of 315 to 400 nm. Suitably the near-UV light may have a wavelength of 375 nm. When the light is in the visible region the wavelength of the light may optionally be 450 to 550 nm, 470 to 500 nm, 480 to 490 nm, or be 488 nm. For photolabile blocking reagents the choice of wavelength should be informed by the photoreactivity of the photolabile moiety, i.e. the wavelength used must lead to cleavage of the photolabile moiety.

[0100] As stated previously the power density of the light needed to remove the desired amount of blocking reagent from the portion of the blocked surface will heavily depend on, particularly, the identity of the blocking reagent. Photolabile blocking reagents will, as a general rule, require lower power densities due to their inherent photoinstability.

[0101] In some embodiments step iii comprises irradiating the portion of the blocked surface with light of a power density of greater than 1 KWrrr2. When the blocked surface comprises a photolabile reagent suitably the power density is 1 KWrrr2to 5 GWirr2

[0102] When the blocked surface comprises a photolabile reagent and near UV light is used in step iii, the power density is suitably 1 KWm’2to 2 MWm’2Suitably the power density is 10 KWITT2to 1 MWm-2. Suitably the power density is about 500 KWm-2. Such power densities are suitable for effective and rapid removal of blocking reagents such as PC-PMOXA-NHS carbonate ester.

[0103] When the blocked surface comprises a photolabile reagent and visible light is used in step iii, suitably the power density is 1 to 5 GWm’2. Suitably the power density is 2 to 4 GWm’2. Suitably the power density is about 2.5 GWm’2. Such power densities are suitable for effective and rapid removal of most photolabile blocking reagents.

[0104] When the blocking reagent does not comprise a photolabile reagent and visible light is used during step iii, suitably the power density is 1 to 12.5 GWm’2. Suitably the power density is 2 to 10 GWm’2. Such power densities are suitable for effective and rapid removal of most blocking reagents including non-photolabile blocking reagents.

[0105] The duration could theoretically be of any length however in general the irradiation of the portion of the blocked surface should be at least 10 seconds for a non-photolabile blocking reagent, suitably 30 to 180 seconds, or 60 to 120 seconds as this enables more rapid throughput of samples. For photolabile blocking reagents the duration can be shorter as these reagents more readily detach from the surface under the right irradiation conditions. For example for photolabile blocking reagents the duration is suitably at least 0.1 s, 0.1 to 30 seconds, 1 to 10 seconds, or 5 to 10 seconds.

[0106] The duration of the irradiation is closely tied to the power density and wavelength of light used. Together they define the total energy imparted to the portion of the blocked surface (Jrrr2) which directly correlates with the amount of blocking reagent that is removed.

[0107] In some embodiments step iii comprises irradiating the portion of the blocked surface with at least 200 KJm’2energy. When the blocking reagent comprises a photolabile reagent, suitably the portion of the blocked surface is irradiated with 200 KJm-2to 150 GJm-2energy.

[0108] When the blocked surface comprises a photolabile reagent and near UV light is used in step iii, the blocked surface is suitably irradiated with 200 KJm’2to 250 MJm’2energy. Suitably the blocked surface is irradiated with 1 to 100 MJm’2energy. Suitably the blocked surface is irradiated with about 10 MJm-2energy. Such energies are suitable for effective of blocking reagents such as PC-PMOXA-NHS carbonate ester.

[0109] When the blocked surface comprises a photolabile reagent and visible light is used in step iii, suitably the portion of the blocked surface is irradiated with 1 to 150 GJrrr2energy. Suitably the blocked surface is irradiated with 5 to 100 GJnr2energy. Suitably the portion of the blocked surface is irradiated with 10 to 50 gJnr2energy. Suitably the blocked surface is irradiated with about 25 GJnr2energy. Such energies are suitable for effective of most photolabile blocking reagents.

[0110] When the blocking reagent does not comprise a photolabile reagent and visible light is used during step iii, suitably the portion of the blocked surface is irradiated with 60 to 4500 GJrrr2energy. Suitably the portion of the blocked surface is irradiated with 500 to 3000 GJnr2energy. Suitably the portion of the blocked surface is irradiated with about 1500 GJnr2energy. Such energies are suitable for effective of most blocking reagents including non-photolabile blocking reagents.

[0111] The invention also provides a method for performing a mass photometry measurement of an object comprising the method of generating a binding surface as described above followed by: iv. adding the object in solution to the binding surface; and v. making a mass photometry measurement.

[0112] Adding the object in solution to the binding surface can be accomplished by any suitable method known by the skilled person. The object in solution may be manually added to the binding surface by a user, or this could be automated using a machine. Where the base surface is the surface of a solid immersion lens, adding the object in solution to the binding surface may comprise dipping the solid immersion lens into the solution. Where the base surface is the surface of a flow cell the object in solution may be flowed over the binding surface.

[0113] The method may be adapted to a method of performing n mass photometry measurements of an object or objects by following steps i. to v. followed by n-1 cycles of steps ii. to v., wherein each iteration of step ii targets a portion of the blocked surface which is spatially distinct from any previously targeted portions of the blocked surface; and wherein the object in solution in each iteration of step iv is independently selected from a set of m objects where m < n.

[0114] This method is advantageous because the binding surface generated in each iteration of step iii. is fresh and has not been exposed to any of the previous objects. Furthermore the surface surrounding the binding surface cannot be affected by accumulation of protein and therefore the mass photometry measurement is unlikely to be impacted by cross contamination. Thus, the mass photometry measurements made using these methods are of greater reliability and accuracy and are not prone to a decrease in binding counts as time proceeds as is observed with traditional mass photometry methodologies.

[0115] The method may be used to obtain multiple mass photometry measurements of the same object, optionally under different conditions such as different solutions, different concentrations, with different amounts of blocking reagent removed, or any other difference. Alternatively, or in addition, the method may be used to obtain mass photometry measurements of different objects using the same base surface without risk of contaminating the binding surface.

[0116] The method may be used to obtain multiple mass photometry measurements of an object at defined time periods, for example at defined time periods after the object is exposed to a change such as addition of a reagent the object interacts with or a change in concentration of the object. n is theoretically limited only by the geometry of the blocked surface and the size of the portions of the blocked surface which are irradiated. In some embodiments n is 384 or less. In some embodiments n is 96 or less. In some embodiments n is 48 or less. These numbers enhance compatibility with standard high-throughput sample preparation with commonly used plates.

[0117] Optionally the method is caried out within a flow cell having a flow defining an upstream direction and a downstream direction. A flow cell allows for greater automation of the methods of the invention. The flow cell may be part of a larger sample handling system capable of delivering solutions comprising objects, buffers, intermediary reagents and / or blocking reagents to the flow cell as well as, optionally, delivering air to the flow cell.

[0118] As shown in Figure 2A, optionally, the portion of the blocked surface targeted in an iteration p+1 of step ii is in the upstream direction from the portion of the blocked surface targeted in iteration p of step ii. In this way the portion of the blocked surface targeted in iteration p+1 of step ii and the resulting binding surface is more likely to be free of contamination by objects or other reagents which were measured in iteration p of the method.

[0119] As shown in Figure 2B, optionally, the portion of the blocked surface targeted in an iteration q+1 of step ii is offset from the portion of the blocked surface targeted in iteration q of step ii in a direction perpendicular to the flow. This also serves to reduce the chance of contamination by previously measured objects or reagents. As shown in Figure 2C, optionally, the portion of the blocked surface targeted in an iteration s+1 of step ii is offset from the portion of the blocked surface targeted in iteration s of step ii in directions both perpendicular to the flow and parallel with the flow direction. This also serves to reduce the chance of contamination by previously measured objects or reagents.

[0120] As shown in Figure 2D, optionally, the portion of the blocked surface targeted in an iteration t+1 of step ii is offset from the portion of the blocked surface targeted in iteration t of step ii in a direction perpendicular to the flow; and the portion of the blocked surface targeted in an iteration t+o of step ii is offset from the portion of the blocked surface targeted in iteration t of step ii in a direction parallel to the flow. This also serves to reduce the chance of contamination by previously measured objects or reagents.

[0121] The invention also provides a glass surface comprising a binding surface for an object in a solution, wherein the glass surface comprises: i. a blocked surface comprising a base surface and at least one blocking reagent; ii. an unblocked surface wherein a portion of the blocked surface has been irradiated with a light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface; wherein the binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface.

[0122] Optionally the glass surface is the surface of a glass slide, the surface of a solid immersion lens, or the surface of a flow cell.

[0123] The invention also provides a flow cell wherein substantially all of the fluid contacting surfaces of the flow cell comprise a layer of blocking reagent, wherein the flow cell comprises a base surface for binding an object in a solution, wherein i. the base surface is blocked by at least one blocking reagent; and ii. wherein a portion of the blocked surface can be irradiated with a light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface; and wherein the binding rate of the object to the blocked surface is less than the binding rate of the object to the binding surface. In some embodiments, the flow cell may be made from a material which can acts to block or prevent an object binding to at least a portion of the flow cell.

[0124] In some embodiments the binding rate or net binding rate of the object to the blocked surface is less than 20% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 5% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 2% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 1 % of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is at least 0.1% and less than 20% of the binding rate of the object to the base surface.

[0125] The invention also provides a method of generating a binding surface for objects in a solution comprising the steps of: i. adding a blocking reagent to a base surface to form a blocked surface; ii. targeting of a portion of the blocked surface with a light source; iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface; wherein the binding rate of the objects to the blocked surface is less than the binding rate of the objects to the base surface.

[0126] In some embodiments the binding rate or net binding rate of the object to the blocked surface is less than 20% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 10% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 5% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 2% of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is less than 1 % of the binding rate of the object to the base surface. Suitably the binding rate or net binding rate of the object to the blocked surface is at least 0.1% and less than 10% of the binding rate of the object to the base surface. Examples

[0127] Example 1 - Repeated Mass photometry measurement leads to accumulation of objects.

[0128] Figures 3a and 3b show the effect of sample buildup under standard mass photometry conditions when reusing a surface. Bovine IgG, 1.2 nM concentration in PBS was flowed continuously at 100 mL / min in a flow cell comprising a glass surface coated with a trialkoxyalkylaminosilane. Binding behaviour is checked every 10 measurement cycles (equivalent to 10 mass photometry measurements). After 20 cycles there is a change in binding behaviour associated with protein accumulating on the surface. All measurements were in the same region of the cell. The laser was on only for finding focus and taking measurements. Unbinding:binding ratio is the protein unbinding counts divided by protein binding counts.

[0129] Example 2 - Blocking of surface with PEG

[0130] To a glass slide pre-functionalised with a trialkoxyalkylaminosilane was added 100 pL PEG solution containing PEG (5000 Da) at 45 mg / mL and 8.4 mg / mL sodium bicarbonate, pH 9.5. A second trialkoxyalkylaminosilane functionalised slide was used to sandwich the PEG solution. The slides were incubated for roughly 16 hours at room temperature and then separated and washed with milli-Q water.

[0131] Example 3 - Use of PEG as a blocking reagent

[0132] Figure 4 shows the detected binding events from 96 sequential mass photometry measurements in the same flow cell. The sample was bovine IgG (1.7 nM in PBS) and was measured on a TwoMP mass photometer. A flow system was used to deliver the sample to a flowcell (100 uL / minute), This was coupled to a glass coverslip that was coated with a trialkoxyalkylaminosilane and PEG (5000kDa) as described in Example 2. The surface was exposed to the following sequence PBS (100 uL / minute, 11 .25 s), PBS (300 uL / minute, 11 .25 s), Air (60 s) during which time a surface was unblocked to allow protein binding by illuminating a 10x4 pm area of the flow cell measurement surface with -500 mW laser power (488 nm), PBS (300 uL / minute, 11.25 s), PBS (100 uL / minute, 11.25 s). The number of binding counts is for protein detected in the 130-170 kDa mass range. Each measurement was taken using a freshly unblocked surface in a spatially distinct region of the flowcell measurement surface. The data for the blocked surface was obtained by taking a standard mass photometry T1 measurement without performing an unblocking step. The data for the blocked surface shows net binding (total binding counts - total unbinding counts).

[0133] Example 4 - Blocking of surface with BSA

[0134] A glass slide pre-functionalised with a trialkoxyalkylaminosilane was exposed to 100 cycles of the following:

[0135] 30 nM BSA (100 uL / minute, 5 minutes),

[0136] Ethanol / TAPs 70:30 with 1 mM EDTA (100 uL / minute, 1 minutes).

[0137] Example 5 - Use of BSA as a blocking reagent

[0138] Figure 5 shows the detected binding events from 96 sequential mass photometry measurements of IgG (1.2 nM in PBS) in the same flow cell using a TwoMP mass photometer. A flow system was used to deliver the sample to a flowcell (100 uL / min), which was coupled to a glass slide prepared as in Example 4. The surface was exposed to the following sequence PBS (100 uL / minute, 30 s), milli-Q (300 uL / minute, 30 s), ethanol / TAPs (70:30 with 1 mM EDTA) (300 uL / minute, 30 s) Air (60 s) during which time a surface was unblocked to allow protein binding by illuminating a 10x4 pm area of the flow cell measurement surface with -500 mW laser power (488 nm), ethanol / TAPs (70:30 with 1 mM EDTA) (300 uL / minute, 30 s), milli- Q (300 uL / minute, 30 s), PBS (100 uL / minute, 30 s). The number of binding counts is protein detected in the 130-170 kDa mass range. Each measurement was taken using a freshly unblocked surface in a spatially distinct region of the flowcell measurement surface.

[0139] Example 6 - Blocking of surface with photolabile PEG

[0140] To a glass slide pre-functionalised with a trialkoxyalkylaminosilane was added 100 pL PEG solution containing photocleavable azido-PEG11-NHS carbonate ester at 33 mg / mL and 8.4 mg / mL sodium bicarbonate, pH 9.5 (prepared by dilution of 208 mg / mL solution of PC Azido- PEG11-NHS carbonate ester in DMSO).

[0141] A second trialkoxyalkylaminosilane functionalised slide was used to sandwich the photolabile PEG solution. The slides were incubated for 2 hours at room temperature and then separated and washed with milli-Q water. Care was taken to minimise exposure of the slides, solutions, or reagents to light. Example 7 - Use of photolabile-PEG as a blocking reagent

[0142] Figure 6 shows the detected binding events from 96 sequential mass photometry measurements of Bovine IgG (10 nM in PBS) in the same flow cell using a TwoMP mass photometer. A flow system was used to deliver the sample to a flowcell (100 uL / minute), which was coupled to a glass coverslip that was coated with a trialkoxyalkylaminosilane and PC Azido-PEG11-NHS carbonate ester. A surface was unblocked to allow protein binding by illuminating a 17x12 pm area for 120 seconds at 500 mW laser power (488 nm), with PBS in the flow cell (flow rate 300 uL / minute). The number of binding counts is protein detected in the 130-170 kDa mass range. The data for the blocked surface was obtained by taking a standard mass photometry measurement with 50 mW laser power and without performing an unblocking step. The data for the blocked surface shows net binding (total binding counts - total unbinding counts).

[0143] Example 8 - Variable removal of blocking reagents

[0144] Figures 7a and 7b show the time dependence of removal of [a] PC Azido-PEG11-NHS carbonate ester in a 17 x 12 pm2area in the presence of PBS and [b] PEG (5000 kDa) in a 11 x 4 pm2area using the sequence PBS (11.25 s @ 100 uL / minute then 11.25 s @300 uL / minute) followed by air (duration shown in figure) and then PBS (11.25 s @ 300 uL / minute then 11.25 s @100 uL / minute) . In both experiments surfaces were irradiated with an 488 nm laser with 500 mW laser power. The sample used was bovine IgG (1.66 nM flowing at 100 uL / minute). The duration of surface illumination during the main blocking step ([a] PBS, [b] air) is given on the x-axis prior.

[0145] This is followed by a standard mass photometry measurement. Counts rise as the blocking layer is progressively removed. PC Azido-PEG11-NHS carbonate ester reaches a stable number of counts after 15 seconds of exposure to the unblocking laser suggesting that the blocking layer is fully removed by this time, although it likely reaches this state more quickly. PEG takes roughly 120 seconds of exposure in air to reach a stable number of counts at this light intensity.

[0146] Example 9 - Blocking of surface with photocleavable polymethyloxazoline-NHS carbonate ester (PC-PMOXA50-NHS carbonate ester) To a glass slide pre-functionalised with a trialkoxyalkylaminosilane was added 100 pL PMOXA solution containing PC-PMOXA-50-NHS carbonate ester at 125 mg / mL, 6.3 mg / mL sodium bicarbonate, 2.7 mg / ml sodium carbonate, pH 9.5.

[0147] A second trialkoxyalkylaminosilane functionalised slide was used to sandwich the PMOXA solution. The slides were incubated for roughly 2 hours at room temperature and then separated and washed with PBS then milli-Q water.

[0148] Example 10 - Use of PMOXA as a blocking reagent

[0149] Figure 8 shows the detected binding events from 167 sequential mass photometry measurements of an ssRNA ladder sample. Counts are shown for the 1000 bases peak only. Measurements were taken on an instrument optically similar to a TwoMP Mass Photometer. The main mass photometry laser was at 520 nm. A flow system was used to deliver the sample to a flowcell (100 pL / minute), which was coupled to a glass coverslip that was coated with a trialkoxyalkylaminosilane and PC-PMOXA50-NHS carbonate ester. A surface was unblocked to allow RNA to bind by illuminating an approximately 25 pm circular area with a laser of wavelength 375 nm for 10 seconds at 1 mW laser power. This area overlapped with the mass photometer field of view with deionised water flowing over the sample at a flow rate of 300 pL / minute . The number of binding counts is the amount of events detected in the 750-1250 bases range. The data for the blocked surface was obtained by taking a standard mass photometry measurement with 200 mW laser power and without performing an unblocking step. The data for the blocked surface shows net binding (total binding counts - total unbinding counts).

[0150] Example 11 - Blocking of surface with photolabile PEG and cloud point functionalisation

[0151] To a glass slide pre-functionalised with a 3-aminopropyldiisopropylethoxysilane was added 100 pL PC-PEG23 solution containing PC-PEG23 at 100 mg / mL, 6.3 mg / mL sodium bicarbonate, 2.7 mg / ml sodium carbonate and 70 mg / ml potassium sulphate, pH 9.5.

[0152] Functionalisation takes place at cloud point because of an increased PEG and salt concentration compared to Example 2, such that the solution is close to the saturation point. A second 3-aminopropylmethyldiethoxysilane functionalised slide was used to sandwich the PC-PEG23 solution. The slides were incubated for roughly 2 hours at room temperature and then separated and washed with PBS then milli-Q water.

[0153] Example 12 - Use of photolabile PEG functionalised at cloud point as a blocking reagent

[0154] Figure 9 shows mass photometry histograms showing an increase in binding counts as a result of unblocking a pegylated surface. A bovine IgG sample was measured on a custom mass photometer optically similar to a twoMP mass photometer instrument. The main mass photometry laser was a 520 nm laser. A flow system was used to deliver the sample to a flowcell (100 pL / minute), which was coupled to a glass coverslip that was coated with 3- aminopropyldiisopropylethoxysilane and PC Azido-PEG23-NHS carbonate ester, which was functionalised at cloud point.

[0155] A surface was unblocked to allow sample to bind by illuminating an approximately 25 pm circular area with a laser of wavelength 375 nm for 40 seconds at 1 mW laser power. This area overlapped with the mass photometer field of view. During the illumination the medium above the surface oscillated twice between deionised water (300 uL / min for 10s) and air (10s). The data for the blocked surface was obtained by taking a standard mass photometry measurement with 200 mW laser power and without performing an unblocking step. The data for the blocked surface shows net binding (total binding counts - total unbinding counts).

[0156] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0157] “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.

[0158] 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.

[0159] 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 method of generating a binding surface for an object in a solution comprising the steps of: i. obtaining a blocked surface wherein the blocked surface comprises a base surface and at least one blocking reagent; ii. targeting of a portion of the blocked surface with a light source; and iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the binding rate of the object to the blocked surface is less than the binding rate of the object to the base surface.

2. The method according to claim 1 wherein the binding rate of the object to the blocked surface is less than 20% the binding rate of the object to the base surface.

3. The method according to claim 1 or claim 2 wherein the base surface comprises a glass surface.

4. The method according to claim 3 wherein the glass surface is the surface of a glass slide, the surface of a solid immersion lens, or the surface of a flow cell.

5. The method according to claim 3 or claim 4 wherein the base surface further comprises a layer of an intermediary reagent configured to form an interface between the glass surface and the blocking reagent, optionally wherein the layer of intermediary reagent is not substantially removed by step iii.

6. The method of claim 5 wherein the intermediary reagent comprises an amine moiety, optionally the intermediary reagent is a compound according to Formula A:Formula A wherein n is 2-6 and R1, R2, and R3are independently selected from Ci-Ce branched or unbranched alkyl or alkoxy, optionally wherein R1, R2, and R3are all methoxy or all ethoxy.

7. The method of any preceding claim wherein the blocking reagent is selected from bovine serum albumin, polyethylene-glycol, or polyoxazoline.

8. The method according to claim 7, wherein during or immediately following step iii the space immediately above the portion of the blocked surface comprises air.

9. The method according to one of claims 1 to 8, wherein visible light is used in step iii and: the power density is 1 to 5 GWm-2, more preferably the power density is 2 to 4 GWm-2, most preferably the power density is about 2.5 GWm-2; the portion of the blocked surface is irradiated for at least 10 seconds, preferably 30 to 180 seconds, more preferably 60 to 120 seconds; or the portion of the blocked surface is irradiated with 60 to 4500 GJnr2energy, preferably 500 to 3000 GJnr2energy, more preferably about 1500 GJnr2energy.

10. The method of one of claims 1 to 7 wherein the blocking reagent comprises a photolabile reagent, preferably wherein the photolabile reagent is a photolabile polyoxazoline or photolabile polyethylene glycol, more preferably the photolabile polyethylene glycol is a PC-Azido-PEG-NHS carbonate ester or PC-Azido-PEG-PFP ester, more preferably the PC-Azido-PEG-NHS carbonate ester is PC Azido-PEG11-NHS carbonate ester or PC Azido-PEG23-NHS carbonate ester, more preferably the photolabile polyoxazoline is photocleavable polymethyloxazoline, more preferably the photocleavable polymethoxazoline is a PC-PMOXA-NHS carbonate ester, more preferably the PC- PMOXA-NHS carbonate ester is PC-PMOXA50-NHS carbonate ester.

11. The method of claim 10 wherein during step iii the space immediately above the portion of the blocked surface comprises a liquid medium.

12. The method claim 10 or 11 , wherein step iii comprises irradiating the portion of the blocked surface with visible light or with near-UV light.

13. The method according to claim 12 wherein the visible light has a wavelength between 450 and 550 nm, preferably between 470 and 500 nm, more preferably between 480 and 490 nm, most preferably 488 nm, or the near-UV light has a wavelength of 315 to 400 nm, more preferably 375 nm.

14. The method according to any one of claims 10 to 13 wherein step iii comprises irradiating the portion of the blocked surface with light of a power density of greater than 1 KWm-2, optionally 1 KWm-2to 5 GWm-2.

15. The method according to claim 14, wherein near-UV light is used in step iii and: the power density is 1 KWm’2to 2 MWm’2, preferably the power density is 10 KWm’2to 1 MWm-2, more preferably the power density is about 500 KWm-2; the portion of the blocked surface is irradiated for at least 0.1 seconds, preferably 0.1 to 30 seconds, 1 to 10 seconds, or 5 to 10 seconds; or the portion of the blocked surface is irradiated with 200 KJm’2to 250 MJm’2energy, preferably 1 to 100 MJm-2energy, more preferably about 10 MJm-2energy.

16. The method according to claim 14, wherein visible light is used in step iii and: the power density is 1 to 5 GWm-2, more preferably the power density is 2 to 4 GWm-2, most preferably the power density is about 2.5 GWm-2; the portion of the blocked surface is irradiated for at least 0.1 seconds, preferably 0.1 to 30 seconds, 1 to 10 seconds, or 5 to 10 seconds; or the portion of the blocked surface is irradiated with 5 to 100 GJm-2energy preferably 10 to 50 gJm’2energy more preferably about 25 GJm-2energy.

17. The method according to any preceding claim wherein the object is a macromolecule such as a protein, a complex of proteins, or nucleic acids.

18. A method of performing a mass photometry measurement of an object in solution comprising the method according to any preceding claim and: iv. adding the object in solution to the binding surface; andv. making a mass photometry measurement.

19. The method of claim 18, wherein step iv. occurs before step ii. or before step iii.

20. A method of performing n mass photometry measurements of an object or objects comprising the method according to claim 18 or 19 followed by n-1 cycles of steps ii-v; wherein each iteration of step ii targets a portion of the blocked surface which is spatially distinct from any previously targeted portions of the blocked surface; and wherein the object in solution in each iteration of step iv is independently selected from a set of m objects where m < n.21 . The method of claim 20 wherein n is 384 or less, 96 or less or 48 or less.

22. The method of claim 20 or 21 wherein the method is carried out within a flow cell having a flow defining an upstream direction and a downstream direction.

23. The method of claim 22 wherein the portion of the blocked surface targeted in an iteration p+1 of step ii is in the upstream direction from the portion of the blocked surface targeted in iteration p of step ii.

24. The method of claim 22 or 23 wherein the portion of the blocked surface targeted in an iteration q+ 1 of step ii is offset from the portion of the blocked surface targeted in iteration q of step ii in a direction perpendicular to the flow.

25. The method of any one of claims 22 to 24, wherein the portion of the blocked surface targeted in an iteration s+1 of step ii is offset from the portion of the blocked surface targeted in iteration s of step ii in directions both perpendicular to the flow and parallel with the flow direction.

26. The method of any one of claims 22 to 24 wherein the portion of the blocked surface targeted in an iteration t+1 of step ii is offset from the portion of the blocked surface targeted in iteration t of step ii in a direction perpendicular to the flow; and the portion of the blocked surface targeted in an iteration t+o of step ii is offset from the portion of the blocked surface targeted in iteration t of step ii in a direction parallel to the flow.

27. A method of generating a binding surface for an object in a solution comprising the steps of:i. adding a blocking reagent to a base surface to form a blocked surface; ii. targeting of a portion of the blocked surface with a light source; iii. irradiating the portion of the blocked surface with the light source under conditions which cause a quantity of the blocking reagent bound to the portion of the blocked surface to be removed, thus creating a binding surface on the base surface; wherein the binding rate of the object to the blocked surface is less than the binding rate of the object to the base surface.

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