WO2020009642A1

WO2020009642A1 - A real-time online biosensor system for combined uv-light detection and nanoplasmonic sensing using localized surface plasmon resonance

Info

Publication number: WO2020009642A1
Application number: PCT/SE2019/050626
Authority: WO
Inventors: Daniel Aili; Ingemar LUNDSTRÖM; Erik MARTINSSON
Original assignee: Arguseye Ab
Priority date: 2018-07-03
Filing date: 2019-06-27
Publication date: 2020-01-09
Also published as: SE1850840A1; EP3818360A1

Abstract

The present invention relates to an online biosensor system and method for combined real-time UV-light monitoring and nanoplasmonic sensing based on LSPR. The biosensor system comprises a sensor module, a light source module, optical fiber module and a detector module. The sensor module comprises at least one sensor chip which is contact with a sample. A first sensor chip of the sensor module comprises a surface and plasmonic nanoparticles. The plasmonic nanoparticles are immobilized on said surface or alternatively on an optional substrate con- figured above said surface. The nanoparticles capture molecules, such as the antibodies, and said molecules have specificity for one or more analytes. The light source module emits a first light in the UV light region as well as a second light in the visible region or in the near-infrared region. The optical fiber module comprises at least one optical fiber which is connected to the sensor module, light source module and the detector module. The optical fiber module passes light from the light source module to the sensor module. Moreover, the optical fiber module also passes reflected or transmitted light from the sensor module directly or indirectly to the detector module. The detector module detects reflected or transmitted UV light and LSPR shift from said sensor module. …

Description

A REAL-TIME ONLINE BIOSENSOR SYSTEM FOR COMBINED UV-LIGHT DE- TECTION AND NANOPLASMONIC SENSING USING LOCALIZED SURFACE PLASMON RESONANCE

Technical field

The present invention relates to a real-time online biosensor system and method for UV-light spectrophotometry and nanoplasmonic sensing using localized sur- face plasmon resonance (LSPR).

Background art

[0001 ] Biologies are macromolecular drugs derived from biological sources such as organs and tissues, microorganisms, animal fluids, or genetically modified cells and organisms. Biologies are also referred to as biopharmaceuticals, biologic(al) medical products and biologicals in the art. Currently, they are rapidly transforming both the pharma industry and the healthcare sector as they provide novel means for treating a wide range of diseases where no options had been made available before. In 2016, 8 out of 10 highest selling drugs were biologies [1 ] However, the production of biologies is extremely challenging and requires very sophisticated manufacturing facilities that are immensely costly to operate. The drugs conse- quently tend to be very expensive and practically unaffordable to a large part of the global population and pose a large burden to already strained healthcare budgets. As an example, a one-year treatment with a monoclonal therapeutic anti- body can cost as much as $400 000 per patient [2] Reducing these costs by streamlining process development, and increasing productivity and yield in the production processes would greatly facilitate for the healthcare system to take on these new drugs. Thus, there is an increasing pressure from authorities on the biopharma industry to reduce the price of biologies.

[0002] To stay competitive and reduce the costs, there is a need to increase productivity and yield in the production of biologies. This requires new advanced process analytical technologies (PAT) that enable online monitoring of critical qual- ity attributes (CQA), such as target product titer, presence of adverse forms of the target as well as other critical impurities [3] Without real-time online monitoring along the process train the efficiency of continuous processing cannot be reached. Continuous processing has several serious advantages: it requires smaller plants with lesser space volumes and the process generate fewer by-products and re- quires less energy and personnel, as well as significantly lower investments in process equipment and process installation [4] The biotech and biopharma indus- try is thus actively searching for new disruptive technologies that enable sensitive and specific online real-time monitoring of biomolecular analytes. Despite large efforts by both the research community and industry to develop such techniques little progress has been reported.

[0003] Despite large efforts in the industry to develop techniques for online mon- itoring based on e.g. spectroscopic techniques (UV-vis, NIR, MIR, Raman, fluo- rescence), analytical chromatography (HPLC, UPLC), and biosensors (surface plasmon resonance, electrochemical), progress has been limited so far [5] As of today, there are no existing technologies on the market that offers real-time online detection of CQA in the production line. Quality controls are routinely performed offline in quality control (QC) labs using various analytical techniques such as mass spectrometry, high performance liquid chromatography (HPLC) and immu- noassays (e.g. ELISA). Such systems are provided by large companies such as Bruker, Thermo Fisher and Pall Corporation, but these techniques are not suitable for online integration.

[0004] Mass spectrometers (MS) capable of measuring the molecular weight of macromolecules are bulky and expensive equipment. Albeit sample handling can be automatized MS cannot provide information in real-time and would be very dif- ficult to integrate for online data sampling. Likewise, immunoassays require exten- sive sample treatment that is not compatible with the need for rapid online detec- tion. HPLC on the other hand provide means for online detection, but is expensive, time consuming and provide limited information about the species in the sample and thus requires extensive calibration. Numerous companies develop biosensors capable of label-free real-time interaction analysis, such as the Biacore (GE Healthcare), Q-Sense (Biolin Scientific), and Octet (Pall Forte Bio), but none of these technologies have proven apt for online integration. This is mainly due to inherent technical limitations (e.g. large sensitivity to temperature fluctuations, large sensing depth that result in large background signal, and difficulties with sensor multiplexing) that result in complex and expensive instrument designs that are not suitable for continuous online detection. The research community, biotech and biopharma industries are hence actively searching for new disruptive technol- ogies that can enable sensitive and specific online real-time monitoring of bio- molecular analytes.

[0005] Recently a strategy based on localized surface plasmon resonance (LSPR) combined with fiber optics was demonstrated in PCT/EP2016/071720 to fulfill the criteria for continuous online monitoring of CQA.

[0006] The nanoplasmonic sensing technique which is used in

PCT/EP2016/071720 is based on a transduction mechanism that exploits an opti- cal phenomenon known as localized surface plasmon resonance (LSPR). LSPR occurs in metal nanostructures, such as gold nanoparticles (AuNPs), where light of a particular frequency can induce collective oscillations of electrons in the nano- particle. The LSPR frequency is highly dependent on the local dielectric environ- ment in the local vicinity of the nanoparticles. Small changes in the refractive index caused by e.g. binding of analytes to ligands immobilized on the nanoparticle sur- face alter the LSPR frequency, which can be detected spectrophotometrically. Compared to SPR, LSPR is much less sensitive to temperature fluctuations and changes in the background and requires significantly less complex optical setups. Benchtop biosensors based on LSPR are used in laboratory settings for sensitive detection of biomolecular interactions.

[0007] By combining nanoplasmonic sensing with fiber optics in

PCT/EP2016/071720, a very flexible sensor configuration as well as miniaturized sensing system is provided which shrinks a benchtop biosensor to the tip of an optical fiber. The nanoparticles that generates the signal are integrated in a re- placeable sensor chip (referred to as end-cap) which is connected to an optical fiber and which guides the optical signal from the light source to a detector. The design of the sensor probe ensures a high signal-to-noise ratio of the optical re- sponse and a unique sensor performance. However, when the system in

PCT/EP2016/071720 comprising a single sensor chip (i.e. endcap) connected to an optical fiber is applied in a downstream biologies purification operation, only limited information appears to be provided since there are no means to account for non-specific binding and sensor drift. Hence, there is a need for providing an

LSPR system and method which takes in to account non-specific binding and sen- sor drift.

[0008] Moreover, PCT/EP2016/071720 is silent about enabling online system integration that is capable of simultaneously measuring multiple CQA parameters in real-time with high sensitivity. Additionally, PCT/EP2016/071720 is silent about managing non-specific binding and sensor drift. Hence, there is a need for provid- ing an LSPR system and method which provides simultaneously measuring multi- pie CQA parameters in real-time with high sensitivity. There is a need for LSPR system and method which provides means for monitoring and/or compensating non-specific binding and sensor drift.

[0009] More importantly, PCT/EP2016/071720 is silent about combining LSPR with a different analytical technique such as UV-spectrophotometry. Furthermore, PCT/EP2016/071720 is silent about using a combination of LSPR with a different analytical method for real-time online biosensing. Hence, there is a need for providing an LSPR system and method which enables coupling with another ana- lytical method and thereby provides simultaneously measuring multiple CQA pa- rameters in real-time with high sensitivity.

Object of invention [0010] An object of the invention is to provide a system and method for real-time qualitative and/or quantitative analysis of analytes.

[0011 ] A further object of the invention is to provide a system and method for online analysis of analytes. [0012] A further object of the invention is to provide a system and method for both real-time and online analysis of analytes.

[0013] An object of the invention is to reduce research and development (R&D) and production costs of biologies and other pharmaceuticals. [0014] A further object of the invention is to increase productivity and yield in the production of biologies.

[0015] A further object of the invention is to provide a system and method for online monitoring of critical quality attributes (CQA), such as target product titer, presence of adverse forms of the target as well as other critical impurities. [0016] A further object of the invention is to provide a system and method for real-time monitoring of critical quality attributes (CQA), such as target product titer, presence of adverse forms of the target as well as other critical impurities.

[0017] A further object of the invention is to provide a system and method for online and real-time monitoring of critical quality attributes (CQA), such as target product titer, presence of adverse forms of the target as well as other critical impu- rities.

[0018] A further object of the invention is to provide a system and method with reduced manual handling.

[0019] A further object of the invention is to provide a system and method that enables continuous processing.

[0020] A further object of the invention is to provide a system and method with consistent product quality.

[0021 ] A further object of the invention is to provide a system and method for simultaneously measuring multiple CQA parameters in real-time. [0022] A further object of the invention is to provide a system and method for simultaneously measuring multiple CQA parameters in real-time with high sensitiv- ity.

[0023] A further object of the invention is to provide a LSPR system and method which takes in to account non-specific binding and sensor drift.

[0024] A further object of the invention is to provide a LSPR system and method which provides simultaneously measuring multiple CQA parameters in real-time with high sensitivity.

[0025] A further object of the invention is to provide a sensor module which can be used for samples having a velocity.

[0026] A further object of the invention is to provide a sensor module which can be used for liquid samples.

[0027] A further object of the invention is to provide a system and method for use in online and real-time monitoring of one or more compounds in a sample. [0028] A further object of the invention is to provide a system and method for use in online and real-time monitoring of pharmaceuticals and/or biologies.

[0029] A further object of the invention is to provide a system and method for use in online and real-time monitoring of peptides, proteins, recombinant peptides and proteins, enzymes, substrates of enzymes, antibodies or fragments thereof, antigens of antibodies, carbohydrates, lipids, nucleotides, nucleic acids, hor- mones, vaccines, blood, blood components, allergenics, cells such as somatic cells, gene therapies and/or tissues.

[0030] A further object of the invention is to provide a system and method for use in the production of peptides, proteins, recombinant peptides and proteins, enzymes, substrates of enzymes, antibodies or fragments thereof, antigens of an- tibodies, carbohydrates, lipids, nucleotides, nucleic acids, hormones, vaccines, blood, blood components, allergenics, cells such as somatic cells, gene therapies and/or tissues. Summary of invention

[0031] The objects of the invention are solved by the claims.

[0032] A preferred embodiment of the invention relates to a biosensor system and method for combined real-time UV-light monitoring and nanoplasmonic sens- ing based on localized surface plasmon resonance (LSPR), wherein the system and method comprises:

a. a sensor module comprising at least one sensor chip, wherein each sen- sor chip of the sensor module is a piece of solid material having a prox- imal end and a distal end, wherein the surface of the proximal end is configured to be in contact with a sample,

b. wherein a first sensor chip of said sensor module comprises plasmonic nanoparticles immobilized on said surface of the proximal end or alter- natively immobilized on an optional substrate configured above the sur- face of the proximal end,

c. a light source module configured to emit to the sample (i) a first light in the UV light region, and (ii) a second light in the visible region or in the near-infrared region,

d. an optical fiber module, comprising at least one optical fiber which is di- rectly or indirectly connected to the sensor module, light source module and a detector module,

e. wherein said optical fiber module is configured to pass light from the light source module to said sensor module, and wherein said at least one op- tical fiber is further configured to pass reflected or transmitted light di- rectly or indirectly from said sensor module a detector module, and f. wherein the detector module comprises at least one detector, wherein the detector module is configured for detecting reflected or transmitted UV-light and LSPR shift from said sensor module.

[0033] A biosensor method for combined real-time UV-light monitoring and na- noplasmonic sensing based localized surface plasmon resonance (LSPR) is also provided. [0034] Use of the biosensor system or biosensor method is furthermore provid- ed.

[0035] A flow cell for the biosensor system is provided.

[0036] Further advantageous embodiments are described in the dependent claims.

Brief description of drawings

[0037] The invention is now described, by way of example, with reference to the accompanying drawings, in which:

[0038] Figure 1 illustrates a single reflection detection biosensor system and method.

[0039] Figure 2 illustrates sensor modules of single detection and dual detection biosensor systems and methods. Figure 2a relates to single reflection detection sensor module. Figure 2b relates to single transmission detection sensor module. Figure 2c relates to dual reflection detection sensor module. Figure 2d relates to dual transmission detection sensor module.

[0040] Figure 3 illustrates a dual reflection detection biosensor system and method.

[0041 ] Figure 4 illustrates sensor modules of dual detection biosensor systems and methods. Figures 4a and 4b relates to dual reflection detection sensor mod- ules having different configurations. Figures 4c and 4d relate dual transmission detection sensor modules having different configurations.

[0042] Figure 5 illustrates a quadruple reflection detection biosensor system and method.

[0043] Figure 6 illustrates plasmonic nanoparticles which are immobilized on the surface of sensor chips. Figure 6a, represents a reference sensor chip without capture molecules while figure 6b represents a sensor chip in which capture mole- cules have been attached to the plasmonic nanoparticles. [0044] Figure 7 illustrates a sensor module which comprises a housing which has a single inlet and outlet, an opening for each replaceable sensor chip and an aperture for each optical fiber.

[0045] Figure 8 illustrates the spectral changes that occurs in the UV absorption signal (I) and in the LSPR signal (II) when a protein is introduced in the sensor system.

[0046] Figure 9 illustrates a biosensor system and method having a sensor module comprising four sensor chips, i.e. a quadruple reflection detection biosen- sor system and method. [0047] Figure 10 illustrates a comparison between (a) a prior art technique, (b) an embodiment of the present invention.

[0048] Figure 1 1 illustrates the use of the present invention in the production of biological drugs.

[0049] Figure 12a shows light extinction spectra before (III) and after (IV) addi- tion of BSA.

[0050] Figure 12b shows a zoom-in of the LSPR peak region of the spectra shown in Figure 12a.

[0051 ] Figure 13 shows an experiment where BSA (1 mg/mL in PBS) was in jected (500 pL) into the flow system [0052] Figure 14 shows an experiment with a ligand-analyte interaction.

Description of embodiments

[0053] The problems associated with the prior art techniques has been solved by an online biosensor system and method for combined real-time UV-light moni- toring and nanoplasmonic sensing based on LSPR. The biosensor system 1 com- prises a sensor module 10, a light source module 20 optical fiber module 35 and a detector module 40 as illustrated in figures 1 , 3 and 5. Due to the combination of real-time UV and LSPR sensing, the biosensor system can provide simultaneous information on both total protein concentration and concentration of specific pro- teins (e.g product) in the sample. The biosensor system can utilize the detector for both UV-vis spectroscopy and LSPR sensing and is more compact, robust and cost-effective compared to systems comprising separate detection modules.

[0054] The sensor module 10 comprises at least one sensor chip which is in contact with the sample. A first sensor chip 10A of said sensor module comprises a surface 17, plasmonic nanoparticles 18, and wherein the plasmonic nanoparti- cles are immobilized on said surface or on an optional substrate configured above said surface (see figure 6). In sensor chips which act as reference sensor chips such as the one illustrated in figure 6a, capture molecules 19 are not bound to the nanoparticles 18. However, for sensor chips which are for analyte monitoring and biosensing, capture molecules 19 are attached to the nanoparticles as illustrated in figure 6b. The capture molecules 19“capture”, i.e. bind, the analytes 15 that have affinity for. This is shown in figure 6b in which capture molecules (such as antigens) which have affinity for antibodies have captured said antibodies. Conse- quently, the biosensor system functions as a biological sensor (i.e. biosensor) for detecting analytes.

[0055] The light source module 20 emits a first light in the UV light region as well as a second light in the visible region or in the near-infrared region. The optical fiber module 35 comprises at least one optical fiber 35A which is directly or indi rectly connected to the sensor module 10, light source module 20 and the detector module 40. The optical fiber module 35 passes light from the light source module 20 to the sensor module 10. Moreover, the optical fiber module 35 also passes reflected or transmitted light from the sensor module 10 directly or indirectly to the detector module 40.

[0056] An optical fiber of the optical fiber module is either a single mode or mul- ti-mode optical fiber. Moreover, an optical fiber 35A of the optical fiber module 35 may be a bifurcated optical fiber. The common end 36A of the bifurcated optical fiber is configured to be directly or indirectly connected to the sensor module while the first split end 36B and second split end 36C of the bifurcated optical fiber 35A are configured to be directly or indirectly connected to the light source module 20 and the detector module 40, respectively.

[0057] The light source module 20 may comprises two light sources 21 ,22. The first light source emits a first light in the UV light region while the second light source emits a second light in the visible region and/or in the near-infrared region. The first light source and the second light source may be in the same light source housing or in different light source housings. The light source module 20 may hence have two housings, and the housings may be directly or indirectly connect- ed to each other by e.g. electrical wires or optical fibers. In figures 1 , 3 and 5, only the light source module has a single housing.

[0058] The light source module 20 may comprise a single light source 21 which emits light in both the UV wavelength spectrum and the visible, and/or near- infrared, wavelength spectrum. For example, the single light source 21 may be a xenon lamp emitting light in both the UV and visible spectrum. A single light source 21 emitting light across a broader spectrum has the advantage that it is more ro- bust and efficient.

[0059] The UV-monitoring may be carried out at any wavelength which an ana- lyte of interest absorbs UV light, and preferably at one or more wavelengths at which that absorption is at a maximum. A continuous UV source may be used, or alternatively, the UV light may be generated at a single suitable UV wavelength using a discrete light source. The visible light is selected to be of one or more wavelengths that induce LSPR in plasmonic nanoparticles.

[0060] The biosensor system may further comprise an optical switch 60 which is configured between the optical fiber module 35 and the detector module 40. The optical switch selectively switches the light from the sensor module on-and-off.

The detector of the detector module 40 may be a spectrophotometer.

[0061 ] Moreover, the optical fiber module 35 biosensor may be connected to the light source module 20 either directly or indirectly. The indirect connection is via an optical beam splitter 23. A beamsplitter is an optical component used to split a beam of light into two separate parts. The light can be split by percentage of over- all intensity, wavelength, or polarization state.

[0062] A sensor chip of the sensor module may either have a reflecting surface or a transparent surface. When the first sensor chip comprises a reflecting surface then the detector module is configured so that it detects reflected UV light and LSPR shift from the sensor module. When the first sensor chip comprises a wholly or partially transparent surface, then the detector module is configured so that it detects transmited UV light and LSPR shift from the sensor module.

[0063] The sensor module 10 may be replaceable. Moreover, a sensor chip of the sensor module may also be replaceable. Biosensor systems which have more than one sensor chip may be configured either in parallel or in series, i.e. the sen- sor chips are operated either in parallel or in series.

[0064] The biosensor system may operate with a sensor module 10 having a single sensor chip, i.e. the sensor module only comprises the first sensor chip 10A. Such a system may be referred to as a single detection. If single detection biosensor comprises a reflecting surface, then it may be referred to as a single reflection detection biosensor which is illustrated in figures 1 and 2a. In a single reflection detection biosensor, the optical fiber module 35 comprises a bifurcated first optical fiber 35A which is directly or indirectly connected to the light source module 20, the first sensor chip 10A and the detector module 40. The first optical fiber 35A passes light from the light source module 20 to the first sensor chip 10A. Moreover, the first optical fiber 35A is further configured to pass light from the first sensor chip 10A to the detector module 40. Consequently, the detector module 40 detects reflected UV light and LSPR shift from the first sensor chip.

[0065] However, if the single detection biosensor alternatively comprises a par- tially or wholly transparent surface, then it may be referred to as a single transmis sion detection biosensor which is illustrated in figure 2b. In a single transmission detection biosensor, the optical fiber module 35 comprises a first optical fiber 50A and a further optical fiber 55A. The first optical fiber is directly or indirectly con- nected to the light source module 20 and the first sensor chip 10A. The further op- tical fiber 55 is directly or indirectly connected to the first sensor chip 10A and the detector module 40. The first optical fiber 50A passes light from the light source module 20 to the first sensor chip 10A. The further optical fiber 55 passes trans- mitted light from the first sensor chip 10A to the detector module 40. Consequent- ly, the detector module 40 detects transmitted UV light and LSPR shift from the first sensor chip 10A.

[0066] The biosensor may also operate with a sensor module having two sensor chips 10A, 10B. Such a system may be referred to as a dual detection biosensor.

If the dual detection biosensor comprises a reflecting surface, then it may be re- ferred to as a dual reflection detection biosensor and is illustrated in figure 3 as well as figures 2c, 4a and 4b. In a dual detection, the optical fiber module com prises a first optical fiber 35A and a second optical fiber 35B. The first optical fiber 35A is connected to the light source module 20, first sensor chip 10A and the de tector module 40. The second optical fiber 35B is connected to the light source module 20, second sensor chip 10B and the detector module 40. In preferred em- bodiments, the optical fibers are connected to the detector module 40 via an opti- cal switch 60. The first optical fiber 35A passes the first light and/or second light from the light source module 20 to the first sensor chip 10A, and subsequently, the first optical fiber 35A passes the reflected light from the first sensor chip 10A to the detector module 40. In analogy, the second optical fiber 35B passes the first light and/or second light from the light source module 20 to the second sensor chip 10B, and subsequently, the second optical fiber 35B is further pass light from the second sensor chip 10B to the detector module 40. Consequently, the detector module 40 detects reflected UV light and LSPR shift from the sensor chips.

[0067] The first and second optical fibers 35A,35B may be bifurcated optical fibers in the above mentioned three embodiments of the dual reflection detection biosensor, as illustrated in figure 3. The common ends 36A,37A of the bifurcated first optical fiber and the second optical fiber are connected to the first sensor chip 10A and second sensor chip 10B, respectively. The first split ends 36B, 37B of the bifurcated first and second optical fiber are connected to the light source module 20. The second split ends 36C,36D of the bifurcated first and second optical fibers are connected to the detector module 40. Furthermore, the second split ends 36C,37C of the bifurcated first and second optical fibers may be connected to the detector module 40 via an optical switch 60.

[0068] In a specific embodiment of the dual reflection detection biosensor illus trated in figure 4a, the first optical fiber 35A passes both the first light and second light from the light source module 20 to the first sensor chip 10A, and subsequent- ly, the first optical fiber 35A further passes reflected light from the first sensor chip 10B to the detector module 40. Similarly, the second optical fiber 35B also passes the first light and second light from the light source module 20 to the second sen sor chip 10B, and subsequently, the second optical fiber 35B further passes re flected light from the second sensor chip 10B to the detector module 40. Conse quently, the detector module detects reflected LSPR shift and UV light from each of the first sensor chip 10A and second sensor chip 10B.

[0069] In a further specific embodiment of the dual reflection detection biosensor illustrated in figure 4b, the first optical fiber 35A passes only the first light from the light source module 20 to the first sensor chip 10A, and subsequently, the first op- tical fiber 35A further passes reflected light from the first sensor chip 10B to the detector module 40. Similarly, the second optical fiber 35B only passes the second light from the light source module 20 to the second sensor chip 10B, and subse- quently, the second optical fiber 35B further passes reflected light from the second sensor chip 10B to the detector module 40. Consequently, the detector module detects reflected LSPR shift and UV light from the first sensor chip 10A and sec- ond sensor chip 10B, respectively.

[0070] In further embodiment of each of the above mentioned three dual reflec tion detection biosensors, the biosensor system comprises at least one further sensor chip and therefore also at least one further optical fiber, in other words the number of further optical fibers is equal to the number of further sensor chips. The further optical fibers each pass the first light and/or the second light from the light source module to the each of the further sensor chips, and subsequently pass re- flected light from the sensor chips to the detector module, an example of this em- bodiment is illustrated in figure 5 in which the sensor module comprises four sen sor chips. In this embodiment, the common ends 36A, 37A, 38A, 39A of the bifur- cated first to fourth optical fibers 35A, 35B, 35C, 35D are connected to the first sensor to fourth sensor chips 10A, 10B, 10C, 10D, respectively. The first split ends 36B, 37B, 38B, 39B of the bifurcated first to fourth optical fibers are connected to the light source module 20. The second split ends 36C, 37C, 38C, 39D of the bi- furcated first to fourth optical fibers are connected to the detector module 40. Fur thermore, the second split ends of the optical fibers may be connected to the de tector module 40 via an optical switch 60.

[0071 ] However, if a dual detection biosensor alternatively comprises first sen sor chip 10A and second sensor chips 10B which are partially or wholly transpar ent surface, then it may be referred to as a dual transmission detection biosensor and is illustrated in figure 2d. In a dual transmission detection biosensor, the opti- cal fiber module comprises a first optical fiber 50A, second optical fiber 50B, third optical fiber 55A and a fourth optical fiber 55B. The first optical fiber 50A is con- nected to the light source module 20 and the first sensor chip 10A; hence, the first optical fiber 50A passes the first light and/or second light from the light source module 20 to the first sensor chip. The second optical fiber 50B is connected to the light source module 20 and second sensor chip 10B; hence, the second optical fiber 50B passes the first light and/or second from the light source module 20 to the second sensor chip 10B. If only the first light is passed to one of the chips, then the second light has to be passed to the other chip. The third optical fiber 55A is connected to the first sensor chip 10A and the detector module 40; the third op- tical fiber 55A is configured to pass transmitted light from the first sensor chip 10A to the detector module 40. The fourth optical fiber 55B is connected to the second sensor chip 10B and the detector module 40; hence, the fourth optical fiber 55B passes light from the second sensor chip 10B to the detector module. Conse- quently, the detector module 40 detects transmitted UV light and LSPR shift from the sensor chips.

[0072] In a specific embodiment of the dual transmission detection biosensor illustrated in figure 4c, the first optical fiber 50A passes both the first light and sec- ond light from the light source module 20 to the first sensor chip 10A, and subse quently, the third optical fiber 55A passes transmitted light from the first sensor chip 10B to the detector module 40. Similarly, the second optical fiber 50B also passes the first light and second light from the light source module 20 to the sec ond sensor chip 10B, and subsequently, the fourth optical fiber 55B passes re- flected light from the second sensor chip 10B to the detector module 40. Conse- quently, the detector module detects transmitted LSPR shift and UV light from each of the first sensor chip 10A and second sensor chip 10B.

[0073] In a further specific embodiment of the dual transmission detection bio- sensor illustrated in figure 4d, the first optical fiber 50A passes only the first light from the light source module 20 to the first sensor chip 10A, and subsequently, the third optical fiber 55A passes transmitted light from the first sensor chip 10B to the detector module 40. Similarly, the second optical fiber 55A only passes the second light from the light source module 20 to the second sensor chip 10B, and subse- quently, the fourth optical fiber 55B further passes reflected light from the second sensor chip 10B to the detector module 40. Consequently, the detector module detects transmitted LSPR shift and UV light from the first sensor chip 10A and second sensor chip 10B, respectively. Consequently, the detector module 40 de tects transmitted LSPR shift and UV light from the first sensor chip 10A and sec- ond sensor chip 10B, respectively.

[0074] In the present invention, as already indicated, the nanoparticles are ei ther immobilized on the surface of a sensor chip or alternatively on a substrate (see figure 6). If the nanoparticles are immobilized on a substrate, then the sub- strate is either (i) in contact with the surface of the chip, or (ii) in close proximity to the surface. Close proximity is in the present invention defined as 0.1 mm to 1 mm. The substrate may be removable or adhered to the surface of the chip as a film, e.g. a transparent organic or inorganic film. Moreover, the substrate may be transparent.

[0075] The nanoparticles may be selected from lithographic metal nanostruc- tures, metal nanoparticles and semiconductor nanocrystals or a combination thereof, preferably said nanoparticles are metal nanoparticles selected from gold, silver, aluminum, copper platinum and palladium nanoparticles or combinations thereof, more preferably the nanoparticles have a shape selected from a sphere, rod, disk, triangle, cube, star, plate, prism, ellipse, wire, shell, core-shell, rice, ring, or created by a hole in a metal film, most preferably the nanoparticles have a size of 5-250 nm. The nanoparticles are spherical in figure 6.

[0076] Biosensing using metal nanoparticles is a technique where specificity for a desired target analyte is obtained by attaching capturing molecules 19, i.e. lig- ands, to the surface of the nanoparticles (see figure 6b). A wide range of ligands can be used, such as antibodies or antibody fragments, aptamers, and peptides that either are physisorbed or covalently conjugated to the gold nanoparticles us ing various tailored surface chemistries (ideally gold-thiol chemistry). The sensor system can thus be addressed towards various biomolecular analytes by using the appropriate ligands according to the needs of the user. In the system illustrated in figure 6b, the capturing molecules 18 are e.g. antigens which have affinity for anti- bodies 15

[0077] Hence, in summary, as described above, the biosensor system and method according to the present invention can be realized with one or more sen sor chips either by using reflection or transmission spectrophotometry. [0078] Possibilities to do multiple analyte detection in sensor modules compris- ing a single sensor chip may be employed by for example using protein array strategies.

[0079] The possibility of including at least two sensor chips in the sensor module provides simultaneous detection of several different analytes as well as more ad- vanced sensor fusion options and extraction of a much wider range of information (see figures 1 , 3 and 5). These features have the technical effects of providing bet ter means for optimization and control of a production process (see figure 3 or 10). Hence, some embodiments of the present invention provide a LSPR based system and method which comprises a sensor module having multiple sensor chips wherein each sensor chip has specificity for different analytes, or different ligands for the same analyte, or be utilized to compensate for unspecific effects.

[0080] As already described, each sensor chip is individually addressed by opti- cal fibers for illuminating the plasmonic nanoparticles (such as AuNPs) on the sensor chip and for reflecting or transmitting the signal back to the detector mod ule. Multiplexing is provided to reduce the costs of the system and method and is achieved by using a single detector (such as a spectrophotometer) and light source module that will be combined with an optical switch to address the different sensors chips (see figures 1 , 3 and 5).

[0081 ] The sensor module of the present invention may comprise a housing 70 which has a single inlet 75 and outlet 76, an opening 79 for each replaceable sen- sor chip and an aperture 74 for each optical fiber. An example of such a sensor module is illustrated in figure 7 in which the sensor chips 10A, 10B are configured in openings 79 and where the sensor chips are arranged serial configuration. In alternative embodiments of the invention, the sensor chips may be arranged in parallel configuration. The sensor module is preferably configured to withstand typical system pressure in for example in protein purification unit operations (2-20 MPa). This can be achieved by choosing the most optimal combination of tubing 77, fiber optic connectors 78 and/or configurations for fitting the sensor chip(s) in the sensor module (see figure 7). The inlets 75, outlets 76, openings 79 and/or apertures 74 may be threaded or may have other means for connection to tubes, optical fibers, sensor chips. The housing 70 may comprise sub-housings 70A,70B which are connected at connection point 70C to each other to form the housing 70. The sensor module may be referred to as a flow cell in fluidic systems where the sample is transported to the sensor via flow. The sensor module is configured in fluidic systems to withstand system pressures such as 0-50 MPa, preferably 2-20 MPa. The fluid flow may be continuous.

[0082] The present biosensor system is especially suited to use with a flow cell sensor module as the optical signal can be detected instantaneously and can therefore provide real-time sensing of fluid flowing past the sensor chip(s). For clarification, the flow cell sensor module need not comprise the opening 79 or the aperture 74, but need only be adapted to sense a flow of a fluid past the sensor chip.

[0083] A serial configuration might result in a slight time shift in the signal from each sensor chip, however, this time-shift can be compensated by using one of the sensor chips in the senor module a reference sensor chip being free of captur- ing molecules while the other sensor chip is immobilized with nanoparticles and capturing molecules. The time-shift is compensated by collecting UV and LSPR shift data from a reference sensor chip (I) and the other sensor chip (II), and thereafter compensating the time shift by data processing.

[0084] In the present invention, to enable multiplexing, each sensor chip may be individually addressed using a multimodal 2x1 optical fiber, that is connected to the sensor module using connectors (such as standard connectors of type SMA - SubMiniature version A) and/or sealed using O-rings. Sensor chips may be fabri cated as described in PCT/EP2016/071720. The resulting sensor chips are rela- tively long-term stable (ca 2 month) and can be stored under ambient conditions. The sensor chips may be replaceable in order to enable convenient exchange of individual sensor chips and can therefore be mounted from the back/bottom of the sensor module as illustrated in figure 7. The sensor chips may comprise a sub monolayer of nanoparticles (such as 50 nm gold nanoparticles) covering for ex- ample about 10% of the surface. This renders sufficient signal-to-noise level and refractive index sensitivity.

[0085] As already indicated, each sensor chip may be optically connected to the spectrophotometer via a multimode 2x1 optical fiber that is connected to a light source and a detector as schematically outlined in figures 1 ,3 and 5. Since the gold nanoparticles (such as 50 nm spherical AuNPs) show a plasmon band in the visible wavelength range a power-efficient white light source, such as LED, may be used to illuminate the sensor chip(s). In the preferred embodiments of the in- vention, the same LED is used to illuminate the sensor chip(s) via an optical power splitter (PLC) to avoid the need to calibrate for differences in the light source be- tween the chips. For data acquisition, a compact spectrophotometer (e.g. Ocean optics, Avantes) may be used to record the extinction spectra from the sensor chip(s). To enable cost-effective sensor multiplexing an optical switch may be used to connect the sensor chip(s) to a detector module comprising a single detec tor. A data sampling interval of < 5 seconds is preferably used in order to be able to capture and record fast occurring events. With N=10 sensor chips, this requires that each spectrum must be recorded in less than 500 ms, including the time for switching. A multi-mode MEMS Nx1 optical switch with a switching time of less than 40 ms may be used for this. In addition to being relatively fast, the MEMS based optical switches have low power consumption and long-life time, which will ensure that the system and method according to the present invention will be du rable and energy efficient.

[0086] The sensor signals 80 from the detector 40 may be analyzed in a soft- ware that apply signal processing methods and present the results to the operator 90 of the instrument, as illustrated in figure 9. The graphical user interface 89 may display sensorgrams of the in parallel recorded sensor signals from the multi plexed sensor chips 10A, 10B, 10C, 10D. Sensor signals 80 may be fused and analysed with multivariate data analysis 87 methods to generate data of higher precision based on trend analysis and calibrations. This allows to accurately dis- criminate between real (specific) and unspecific events and to discriminate very similar analytes, such as target molecules and impurities in a bioprocess (e.g. in tact, truncated or aggregated product forms). The sensor signals 80 may also be analysed by pattern recognition methods 88, such as machine learning, to reveal additional information in the multiplexed sensor signals. The software may also be prepared for real-time OPC standard communication with external process auto- mation systems. Such communication prepares the multiplexed sensor module as a product for process industry, especially in bioproduction, as illustrated in figure 1 1 .

[0087] An example of a spectrum derived from a biosensor system according to the present invention is shown in figure 8 which illustrates a spectrum from a sen- sor module with combined UV-monitoring and LSPR detection. Gold nanoparticles give rise to a distinct plasmon peak in the visible region of the spectra (solid line). When a protein is introduced in close proximity of the gold nanoparticle surface the LSPR condition is altered and a spectral shift of the plasmon peak (II) occurs (dashed line). Simultaneously, when a protein is introduced in the sensor, an in- crease in the signal intensity (I) in the UV region is obtained due to light absorption by aromatic side chains in the protein. The Y-axis represents light extinction (arbi trary unit, arb.u) and the X-axis represents wavelength (nanometer, nm).

[0088] In the biosensor system and method exemplified in figure 9, the sensor 10) module comprises four sensor chips wherein: (i) the first sensor chip 10A may comprise plasmonic nanoparticles and capture molecules which are specific for a first compound 101 , (ii) the second sensor chip 10B may comprise plasmonic na noparticles and capture molecules which are specific for a second compound 102, (iii) the third sensor chip 10C may comprise plasmonic nanoparticles and capture molecules which are specific for a third compound 103, and (iv) the fourth sensor chip 10D comprises plasmonic nanoparticles without capture molecules, i.e. the fourth sensor chip 10D is a reference sensor chip. Compounds 101 and 102 may for example be pharmaceutical compounds while compound 103 may be a poten tial adverse form (or impurity) of one of said pharmaceutical products. The biosen- sor system in figure 9 illustrates a quadruple reflection biosensor similar to the du al reflection biosensor illustrated in figure 4c but with two further sensor chips. However, it may alternatively also be configured as quadruple transmission bio- sensor similar to the dual reflection biosensor illustrated in figure 4d but with two further sensor chips. Moreover, the biosensor system may comprise fewer or more sensor chips such as only two chips as in figures 4a-d. Furthermore, the reference sensor chip may be omitted, i.e. a biosensor system with only one sensor chips as in figure 1 is also possible. Finally, the biosensor system and method may have a closed loop control 95, i.e. a control system possessing monitoring feedback.

[0089] The present invention solves several problems associated with prior art techniques. One of the problems with prior art techniques is illustrated in figure 10. Prior art protein purification equipment are typically equipped with an online spec- trophotometer capable of detecting the UV-absorbance caused by proteins nor- mally at 280 nm (UV280 signal). However, this data cannot be utilized to distin guish the product from impurities why fractions must be collected in tubes and analysed in a quality control QC lab (figure 10a). Quality controls are unfortunately performed offline in QC labs using various time consuming and expensive analyti cal techniques such as mass spectrometry, high performance liquid chromatog- raphy (HPLC) and immunoassays (e.g. ELISA). Hence, prior art techniques are disadvantageous because they are neither performed online nor performed in real time. As a contrast, using an online biosensor system according to the present invention which is capable of detecting specific compounds 101 , 102, 103 in real- time in the sample, the intended pharmaceutical products 101 ,102 and potential adverse forms 103 can be readily distinguished (figure 10b). This is vital both for optimizing the production and purification process, and for process automation.

[0090] The present invention can be utilized in many different ways in the pro- duction of biological drugs. One use is exemplified in figure 1 1 which shows a large-scale production process of hormones such as insulin in which the present invention may be utilized. Similar processes can be used for antibodies such as monoclonal antibodies. Insulin is by far the most widely used and produced bio- pharmaceutical and is relatively small for being a biological drug, which puts high demands on the sensitivity of the analytical system and method for quantitative or qualitative analysis. Monoclonal antibodies, on the other hand, are high molecular weight compounds and represent the category of blockbuster drugs that is in creasing most rapidly. In preferred embodiments of the present invention, a set of peptide-binders (affibodies), protein A and antibodies may be employed as captur- ing molecules to evaluate and demonstrate multi-analyte detection. Moreover, means to reduced ambiguity and uncertainty in the sensor signals may be evalu ated by employing several different ligands targeting the same analyte.

[0091 ] Bacteria, such as E. coli, which the biological drug is expressed in, is grown initially in a cell culture 105 and then subjected to fermentation in a bioreac- tor 106 as illustrated in figure 1 1 . The bacterial cells are thereafter disrupted 107 and the biological drugs are harvested and/or purified in several steps by using one or more purification devices 140 such as one or more of chromatography col- umns 140A, centrifuges 140B, crystallization chambers 140C and/or freeze driers 140D. Waste 170 is discarded after each harvesting stage and the biological prod uct 160 is collected after the final formation. The biosensor system and method of the present invention incorporated after each purification device 140 for online and real-time quantitative and/or qualitative analysis the biological product and impuri- ties after each harvesting stage. The incorporation can be carried out by arranging a sensor module 10, 1 10, 210, 310 according to the present invention after one or more of the purification devices as illustrated in figure 1 1 . The sensor modules 10, 1 10, 210, 310 may either be comprised in a single biosensor system (i.e. all mod- ules are connected to one detector, or more preferably, each sensor module is comprised in its own respective biosensor system (i.e. each module is connected to its own detector). The biggest advantages of incorporating the present invention is the reduced production time and production costs.

[0092] The term UV-monitoring is in the present invention defined as qualitative and/or quantitative UV-light analysis. Similarly, the term nanoplasmonic sensing is in the present invention defined as qualitative and/or quantitative LSPR-based analysis.

Examples Two further examples of the present invention are provided below-

[0093] Example 1 - Combined UV280 and LSPR detection

[0094] A combined, single-spectrum measurement of UV-light absorption at 280 nm and refractometric LSPR detection using gold nanoparticles was performed using a bench-top spectrophotometer (Shimadzu UV-2450). A cover-slip glass substrate (VWR International) was cut into a 9 x 20 mm piece so that it would fit into a 10 mm cuvette. The glass substrate was cleaned in a solution containing a 5:1 :1 mixture by volume of Milli-Q water, 30% hydrogen peroxide (Merck KGaA) and 25% ammonia (Merck KGaA) for at least 20 min at 85° C and thoroughly rinsed with Milli-Q water. A layer-by-layer assembly of polyelectrolytes (PEs) was used to immobilize Au-NPs on the glass substrate. Polyelectrolyte solutions of pol- yethylenimine (PEI, Mw 750 000, Sigma-Aldrich), polystyrene sulfonate (PSS, Mw 75 000, Sigma-Aldrich) and polyallylamine hydrochloride (PAH, Mw 56 000, Sig ma-Aldrich) were prepared with a concentration of 2 mg/ml in 0.5 M NaCI (Sigma- Aldrich) aqueous solutions. The substrate was incubated with 100 pi of polyelec- trolyte solutions for 15 min in the order PEI/PSS/PAH/PSS/PAH with a thorough rinsing of Milli-Q water between each deposition. 100 pi of Au-NP solution

(d=50nm, BBI solutions) was deposited onto the glass substrate for 4h and rinsed with Milli-Q water. [0095] A substrate covered with Au-NPs was placed inside a quartz cuvette and

2 ml of PBS was added. A non-treated glass substrate was used as reference. A light extinction spectrum (200-800 nm) was acquired followed by an incubation in Bovine Serum Albumin (BSA, Sigma-Aldrich) at a concentration of 10 mg/mL. A new spectrum was acquired after 30 min of incubation in BSA. Figure 12a shows light extinction spectra before (III) and after (IV) addition of BSA. Y-axis: light ex- tinction (arbitrary unit, arb.u). A measurement with nanoparticles is compared to a measurement without nanoparticles. X-axis: light wavelength (nanometer, nm). Addition of BSA gives an increased intensity at around 280 nm due to light absorp tion of amino acids in the protein and a shift of the plasmon peak at around 530 nm due to protein adsorption to the gold nanoparticles.

[0096] Figure 12b shows a zoom-in of the LSPR peak region of the spectra shown in Figure 12a. Y-axis: light extinction (arbitrary unit, arb.u). A measurement with nanoparticles is compared to a measurement without nanoparticles. X-axis: light wavelength (nanometer, nm).

[0097] Example 2 -UV280 and LSPR detection in a protein purification system (AKTA)

[0098] A dual-spectra measurement of UV-light absorption at 280 nm and re- fractometric LSPR detection using gold nanoparticles was performed in a protein purification system from GE Healthcare (AKTA pure). The UV-signal at 280 nm was obtained from the integrated UV-monitor and the LSPR signal was obtained using a custom-built setup. A flow cell designed for a UV-monitor in another AKTA system (AKTA pilot) was disassembled and modified in order to perform LSPR measurements based on reflected signals. A lens holder inside the flow cell was replaced with a solid plug made by stainless steel with the same dimensions as the original lens holder. Reflection tests showed that no additional polishing steps was needed for the plugs after fabrication.

[0099] Stainless steel plugs were immobilized with Au-NPs (d=50nm, BBI solu- tions) using the same protocol as used for the glass substrates in Example 1 . A non-treated plug was used to obtain a background spectrum to remove the contri bution from the substrate and compensate for the spectral distribution from the light source. A functionalized plug was inserted inside the flow cell and connected in the AKTA system directly after the UV monitor. The common end of a bifurcated optical fiber (BIFORO, 01000 pm, 2 m, Ocean Optics) was connected to the flow cell by an SMA connector and the two other fiber ends were connected to a light source (HL-2000-HP-FHSA, 360-2500 nm, 20 w, Ocean Optics) and a detector (QE65 Pro, Ocean Optics), respectively. The signal was analyzed using a custom- designed LabView software.

[00100] Figure 13 shows an experiment where BSA (1 mg/mL in PBS) was in jected (500 pL) into the flow system. BSA first passes the UV-monitor which re sults in a peak in the UV-signal (V) and then enters into the modified flow cell where it is adsorbed onto the Au-NPs which gives rise to a shift in the plasmon peak position (VI). Y-axis (left): UV-light extinction at 280 nm (arbitrary unit, mAu). Y-axis (right): Change in LSPR peak position (picometer, pm). X-axis: time (sec- onds, s).

[00101] Figure 14 shows an experiment with a ligand-analyte interaction. A IgG- binding ligand (1 pM, Zwt from Affibody,) was injected after 200 seconds, which gives a small response in the UV-signal (VII) and a larger response in the LSPR signal (VIII). A second injection with Immunoglobulin G (IgG, 1 pM, PBS, Sigma- Aldrich) was introduced at 700 seconds with resulted in a large UV response and an increase in the LSPR signal due to binding to the ligand. Y-axis (left): UV-light extinction at 280 nm (arbitrary unit, mAu). Y-axis (right): Change in LSPR peak position (picometer, pm). X-axis: time (seconds, s).

References

1 . L. Craig, New 2016 Data and Statistics for Global Pharmaceutical Products and Projections through 2017, ACS Chem.Neurosci. 2017, 8 (8), 1948

2. A. F. Shaughnessy, B!V!J, Monoclonal antibodies: magic bullets with a hefty price tag, 2012, 345:e8346

3. P. Roch and C-F. Mandenius, On-line monitoring of downstream bioprocesses, Curr. Opin. Chem. Eng., 2016, 14, 1 12-120

4. A.S. Rathore et.al ., Continuous processing for production of biopharmaceuti cals, Prep. Biochem. Biotechnol. 2015, 45 (8) 836-849

5. P. Roch and C-F. Mandenius, On-line monitoring of downstream bioprocesses,

Curr. Opin. Chem. Eng., 2016, 14, 1 12-120

6- https://www.qelifesciences.com/en/re/solutions/bioprocessina/services/testa- innovation-center

7. DiCon Fiberoptics Inc., 2018,

htps://www.diconfiberoptics.com/products/mems 1 x n optical switches mul ti mode.php

Claims

1. Biosensor system (1 ) for combined real-time UV-light monitoring and nano- plasmonic sensing based on localized surface plasmon resonance (LSPR), wherein the system comprises:

a. a sensor module (10) comprising at least one sensor chip, wherein each sensor chip of the sensor module is a piece of solid material having a proximal end and a distal end, wherein the surface (17) of the proximal end is configured to be in contact with a sample,

wherein a first sensor chip (10A) of said sensor module (10) comprises plasmonic nanoparticles (18) immobilized on said surface (17) of the proximal end or alternatively immobilized on an optional substrate con- figured above the surface (17) of the proximal end,

b. a light source module (20) configured to emit to the sample (i) a first light in the UV light region, and (ii) a second light in the visible region or in the near-infrared region,

c. an optical fiber module (35), comprising at least one optical fiber (35A, 50A) which is directly or indirectly connected to the sensor module (10), light source module (20) and a detector module (40),

d. wherein said optical fiber module (35) is configured to pass light from the light source module (20) to said sensor module (10), and wherein said at least one optical fiber (35A, 50A) is further configured to pass re- flected or transmitted light directly or indirectly from said sensor module (10) a detector module (40), and

e. wherein the detector module (40) comprises at least one detector,

wherein the detector module (40) is configured for detecting reflected or transmitted UV-light and LSPR shift from said sensor module (10).

2. Biosensor system (1 ) according to claim 1 , wherein capture molecules (19) are arranged on the plasmonic nanoparticles (18) of at least one of the at least one sensor chips (10A), wherein the capture molecules have the ability to bind to one or more analytes in the sample.

3. Biosensor system (1 ) according to claims 1 or 2, wherein an optical fiber (35A, 50A) of the optical fiber module (35) is a bifurcated optical fiber, wherein the common end (36A) of the bifurcated optical fiber is configured to be directly or indirectly connected to the sensor module (10), and wherein the first split end (36B) and the second split end (36B) of the bifurcated optical fiber are config- ured to be connected to the light source module (20) and the detector module (40), respectively.

4. Biosensor system (1 ) according to claims 1 to 3, wherein the light source mod- ule (20) comprises two light sources (21 , 22) , wherein the first light source (21 ) is configured to emit a first light in the UV light region, and wherein the second light source (22) is configured to emit a second light in the visible region or in the near-infrared region, and wherein the first light source (21 ) and the second light source (22) are configured in the same light source housing or in different light source housings.

5. Biosensor system (1 ) according to claims 2 to 4, wherein the sensor module (10) comprises a plurality of sensor chips, and wherein at least one of the plu- rality of sensor chips does not comprise capture molecules (19) and is a refer- ence sensor chip.

6. Biosensor system (1 ) according to claims 1 to 5, wherein the sensor module (10) is a flow cell sensor module (10).

7. Biosensor system (1 ) according to claim 6, wherein the flow cell sensor module (10) comprises a housing (70) which comprises an inlet (75), an outlet (76), an opening (79) for each sensor chip and an aperture (74) for each optical fiber, preferably the sensor module (10) is replaceable, optionally the sensor model (10) further comprises a connector (78) for each optical fiber wherein said con- nector is configured for connecting an optical fiber to said aperture (74).

8. Biosensor system (1 ) according to claims 1 to 7, wherein said at least one sen- sor chip (10A) is replaceable, preferably said sensor module (10) is replacea- ble.

9. Biosensor system (1 ) according to claims 1 to 8, wherein said at least one sen- sor chip (10A) comprises a substrate, and the nanoparticles (18) are immobi- lized on said substrate, wherein the substrate is in contact with the surface (17) or in close proximity to the surface (17), preferably the substrate is removable, more preferably the substrate is transparent, and most preferably the substrate is a transparent organic or inorganic film.

10. Biosensor system (1 ) according to claims 1 to 9, wherein said nanoparticles (18) are selected from lithographic metal nanostructures, metal nanoparticles (18) and semiconductor nanocrystals or a combination thereof, preferably said nanoparticles (18) are metal nanoparticles (18) selected from gold, silver, alu- minum, copper platinum and palladium nanoparticles (18) or combinations thereof, more preferably the nanoparticles (18) have a shape selected from a sphere, rod, disk, triangle, cube, star, plate, prism, ellipse, wire, shell, core- shell, rice, ring, or created by a hole in a metal film, most preferably the nano- particles (18) have a size of 5-250 nm.

11. Biosensor system (1 ) according to claims 1 to 10, wherein said at least one detector of the detector module (40) is a spectrophotometer.

12. Biosensor system (1 ) according to claims 1 to 11 , wherein the sample to be monitored and sensed has a velocity, preferably said sample is liquid a sample, most preferably the liquid sample is configured to flow through the sensor module (10).

13. Biosensor system (1 ) according to claims 1 to 12, wherein the sample to be monitored and sensed has a velocity, preferably said sample is liquid a sample, most preferably the liquid sample is configured to flow through the sensor module (10).

14. Biosensor method for combined real-time UV-light monitoring and nanoplas- monic sensing based localized surface plasmon resonance (LSPR), wherein the method comprises the steps of:

a. providing a sensor module (10) comprising at least one sensor chip, wherein each sensor chip of the sensor module (10) is a piece of solid material having a proximal end and a distal end, wherein the surface of the proximal end is configured to be in contact with a sample, wherein a first sensor chip (10A) of said sensor module (10) comprises plasmonic nanoparticles (18) immobilized on said surface of the proxi- mal end or alternatively immobilized on an optional substrate arranged above the surface of the proximal end,

b. providing a light source module (20) emitting to the sample (i) a first light in the UV light region, and (ii) a second light in the visible region or in the near-infrared region,

c. providing optical fiber module (35) comprising at least one optical fiber (35A, 50A) which is directly or indirectly connected to the sensor module (10), light source module (20) and a detector module (40),

d. wherein said optical fiber module (35) passes light from the light source module (20) to said sensor module (10), and wherein said at least one optical fiber (35A, 50A) further passes reflected or transmitted light from the sensor module (10) directly or indirectly to a detector module (40), and

e. wherein the detector module (40) comprises at least one detector, and wherein the detector module (40) detects reflected or transmitted UV light and LSPR shift from said sensor module (10).

15. Biosensor method according to claim 14, wherein capture molecules (19) are arranged on the nanoparticles (18) of said at least one sensor chip (10A), pref- erably said capture molecules (19) are arranged on the nanoparticles (18) of all sensor chips except for reference sensor chips, wherein said capture mole- cules (19) have the ability to bind one or more analytes in a sample.

16. Biosensor method according to claim 14 or 15, wherein a sample is provided.

17. Use of the biosensor system (1 ) according to claims 1 to 13 or the biosensor method according to claims 14 to 16 for online and real-time monitoring and bi- osensing of one or more compounds in a sample, preferably said one more compounds is selected from pharmaceuticals and/or biologies, more preferably said one or more compounds are selected from peptides, proteins, recombi- nant peptides and proteins, enzymes, substrates of enzymes, antibodies or fragments thereof, antigens of antibodies, carbohydrates, lipids, nucleotides, nucleic acids, hormones, vaccines, blood, blood components, allergenics, cells such as somatic cells, gene therapies and/or tissues.

18. Use of the biosensor system (1 ) according to the claim 17 for monitoring and biosensing of multiple CQA parameters.

19. Use of the biosensor system (1 ) according to claims 1 to 13 or the biosensor method according to claims 14 to 16 in the production of pharmaceuticals and/or biologies, more preferably in the production of peptides, proteins, re- combinant peptides and proteins, enzymes, substrates of enzymes, antibodies or fragments thereof, antigens of antibodies, carbohydrates, lipids, nucleotides, nucleic acids, hormones, vaccines, blood, blood components, allergenics, cells such as somatic cells, gene therapies and/or tissues.

20. Flow cell sensor module (10) for use in the biosensor system according to

claims 1 to 13 or the biosensor method according to claims 14 to 16, wherein the flow cell sensor module (10) comprises a housing (70) which comprises an inlet (75), an outlet (76), at least one sensor chip (10A), an opening (79) for each sensor chip (10A) and an aperture (74) for each optical fiber (35A, 50A), wherein each sensor chip of the sensor module (10) is a piece of solid material having a proximal end and a distal end, wherein the surface of the proximal end is configured to be in contact with a sample, wherein a first sensor chip (10A) of the sensor module (10) comprises plas- monic nanoparticles (18) immobilized on said surface of the proximal end or immobilized on an optional substrate configured above the surface of the prox- imal end.

21. Flow cell sensor module (10) according to claim 20, wherein each sensor chip of the sensor module (10) comprises a substrate and the nanoparticles (18) are immobilized on the substrate, wherein the substrate is in contact with the sur- face or in close proximity to the surface, preferably the substrate is removable, more preferably the substrate is transparent, and most preferably the substrate is a transparent organic or inorganic film.

22. Flow cell sensor module (10) according to claim 20 or 21 , wherein said nano- particles (18) are selected from lithographic metal nanostructures, metal nano- particles (18) and semiconductor nanocrystals or a combination thereof, pref- erably said nanoparticles (18) are metal nanoparticles (18) selected from gold, silver, aluminum, copper platinum and palladium nanoparticles (18) or combi- nations thereof, more preferably the nanoparticles (18) have a shape selected from a sphere, rod, disk, triangle, cube, star, plate, prism, ellipse, wire, shell, core-shell, rice, ring, or created by a hole in a metal film, most preferably the nanoparticles (18) have a size of 5-250 nm.

23. Flow cell sensor module (10) according to claims 20 to 22, wherein the sensor module (10) is configured to withstand a pressure of 0-50 MPa, preferably 2-20 MPa.