A rapid and sensitive biosensor for measuring plasmin activity in milk

Journal Pre-proof A RAPID AND SENSITIVE BIOSENSOR FOR MEASURING PLASMIN ACTIVITY IN MILK Helen Dacres, Jian Wang, Alisha Anderson, Stephen. C. Trowell

PII:

S0925-4005(19)31340-1

DOI:

https://doi.org/10.1016/j.snb.2019.127141

Reference:

SNB 127141

To appear in: Received Date:

18 April 2019

Revised Date:

16 August 2019

Accepted Date:

12 September 2019

Please cite this article as: Dacres H, Wang J, Anderson A, Trowell SC, A RAPID AND SENSITIVE BIOSENSOR FOR MEASURING PLASMIN ACTIVITY IN MILK, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127141

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A RAPID AND SENSITIVE BIOSENSOR FOR MEASURING PLASMIN ACTIVITY IN MILK.

Helen DacresA*, Jian WangB, Alisha AndersonB and Stephen. C. TrowellB A

CSIRO Health & Biosecurity, Food Innovation Centre, 671 Sneydes Road, Werribee, VIC 3030,

Australia Health & Biosecurity, Black Mountain, Clunies Ross Street, Canberra, ACT 2601, Australia

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BCSIRO

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* Corresponding author:

HIGHLIGHTS

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Tel. no: 0011 61 (0)3 97313235

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Email address: [email protected]

A novel BRET-based biosensor was developed to measure trace levels of plasmin activity in milk



A novel peptide target sequence was designed to confer plasmin specificity and sensitivity

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The plasmin biosensor is fully functional in the complex colloidal matrix of milk

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A detection limit of 0.23 nM was achieved in full fat milk with a 10 minute assay time

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Method is useful for managing plasmin-related quality issues in UHT milk and other dairy products

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

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Abstract Plasmin play an important role in milk spoilage and can cause quality issues in UHT milk and other dairy products. Current methods for measuring plasmin activity in milk have low sensitivity and are too slow to be used in routine testing.

We report a bioluminescence resonance energy

transfer (BRET)-based biosensor that can measure naturally occurring low levels of plasmin activity in milk within a few minutes. The biosensor incorporates a plasmin-specific peptide target sequence flanked by the resonance energy BRET transduction elements: green fluorescent

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protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the peptide linker sequence by human plasmin led to an approximately 87

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% decrease in the starting BRET ratio. Using a 10 minute incubation time, the detection limit for human plasmin was 0.25 nM and 0.86 nM for bovine plasmin in 50 % (v/v) full fat milk with EC50s

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of 5.89 ± 0.12 nM and 5.97 ± 0.59 nM, respectively. These detection limits are below the naturally occurring levels of plasmin reported to occur in raw or processed cow’s milk.

The

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plasmin biosensor therefore has the potential to measure naturally occurring plasmin levels directly in raw and UHT-processed milk samples. The protease biosensor described herein is

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selective and sensitive and retains its function in the complex colloidal matrix of milk. It therefore meets many of the requirements for routine use in production settings. We propose

Keywords

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its use for managing plasmin-related quality issues in UHT milk and other dairy products.

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Protease, Endopeptidase, BRET, Bioluminescence, Dairy, Biosensor, UHT

1. Introduction 2

One of the advantages of biosensing over traditional laboratory analytic methods is that biosensors can be deployed in the field to provide rapid on-the-spot measurement and diagnosis. Examples include electrochemical biosensing of glucose in peripheral blood and pregnancy testing from urine using lateral flow immunosensing. However, achieving robust, user-friendly biosensing in complex biological matrices remains challenging and has only been achieved for a relatively small number of mass-market medical applications [1]. Use of biosensors for food analysis lags well behind their use as medical diagnostics. Reasons may include the variety of Milk exemplifies a

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food matrices to be tested and the cost-sensitivities in the food industry.

challenging food matrix. It is a complex solution containing proteins, fats and sugars and has

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been proposed as a suitably challenging matrix for testing novel biosensors [2]. Indeed, matrix effects from milk samples have hindered the development of commercial sensors for milk sample

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analysis [3]. For this study, we set out to develop a robust, selective and sensitive biosensor to measure commercially relevant trace levels of plasmin activity in milk to demonstrate the viability

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of novel optical biosensors for solving a technically challenging problem in food diagnostics. Cow’s milk is a complex and variable biological matrix.

It is substantially transformed

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into a variety of foods through a number of industrial processes, including heat and enzyme treatment as well as microbial and physical processing. Naturally occurring enzymes in raw milk,

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of which there are several [lipases, alkaline and acid phosphatases, lactoperoxidases, sulphydryl oxidase, xanthine oxidase, superoxide dismutase, lysozyme, ribonuclease [4]], as well as endogenous and bacterial proteases, compound the variability in milk’s processing properties. For example, over time, low levels of heat resistant endogenous proteases digest caseins, releasing bitter peptides that increase viscosity and lead to the formation of a gel. The major

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endogenous protease found in milk is plasmin (EC 3.4.21.7), an alkaline serine protease, which readily hydrolyses milk caseins [5]. Plasmin is resistant to heat treatment, surviving both pasteurisation and commercial ultra-heat treated (UHT) processes [6]. Increasing the time and temperature of UHT treatment to completely inactivate plasmin tends to make the milk unpalatable. Currently therefore, the best way to reliably guarantee the shelf-life of UHT milk is to divert raw milk with higher levels of plasmin away from UHT processing. Unfortunately, current methods for measuring plasmin activity in milk are insufficiently sensitive to provide 3

results in time for practical use. Absent a good test, milk processors are obliged to rely on the historical quality profiles of their milk supply, a suboptimal approach. Raw milk with high plasmin activity can therefore cause problems when used to make UHT milk, or milk powder that is subsequently used to make UHT liquid milk. Current methods for measuring plasmin activity in milk include reverse phase high performance liquid chromatography (RP-HPLC) and a variety of colorimetric or fluorometric assays. RP-HPLC is an accurate and sensitive technique for measuring and characterising

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protease degradation of proteins, including casein, once it has occurred [7]. However, because of its post facto nature, RP-HPLC is not useful for measuring low levels of plasmin activities in

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advance of spoilage occurring. Various other methods to measure plasmin activity have been described [8-12]. Many of these assays are based on hydrolysis of a synthetic chromogenic or

[10, 12].

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fluorogenic substrate, which upon cleavage releases a coloured [8, 9, 11] or fluorescent product Richardson and Pearce (1981) developed a fluorogenic assay using a coumarin

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conjugated peptide (N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amido-4-methyl coumarin) where plasmin releases the fluorescent product 7-amido-4-methyl-coumarin.

Using their

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method Richardson and Pearce (1981) reported a concentration range of plasmin in milk of 0.14 - 0.73 µg/ml (1.65 – 8.53 nM).

Rollema et al. (1983) used a similar approach with the Plasmin cleaves the lysine-

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chromogenic substrate H-D-valyl-L-leucyl-lysyl-4-nitroanilide.

nitroanilide bond, releasing 4-nitroanilide which absorbs light at 405 nm [11]. A drawback of these colorimetric and fluorometric assays is that they tend to suffer interference due to the natural turbidity of milk. As an alternative, Collin et al. (1988) developed an enzyme linked immunosorbent assay (ELISA) to measure plasmin levels in dairy products [13]. Plasmin levels

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measured with ELISA predicted higher levels than the proteolytic activities determined by the fluorometric assay of Richardson and Pearce (1981). This was presumably because ELISA measures the amount rather than the activity of plasmin and there is a complex plasmin inhibitory system in milk [8, 14], under normal conditions this suppresses plasmin activity relative to the mass of plasmin present. This complication limits the value of mass-based assays to infer plasmin activity.

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We previously detected picomolar levels of human thrombin, a serine protease[15], using a bioluminescence resonance energy transfer transduction (BRET) scheme [16].

The BRET

transduction was 50 times more sensitive than an equivalent sensor incorporating fluorescence resonance energy transfer transduction. BRET is characterised by the non-radiative transfer of energy from a bioluminescent donor and a fluorescent acceptor. The use of BRET leads to several advantages over traditional colorimetric and fluorometric assays. Compared to intensity-based measurements, the ratiometric nature of BRET reduces signal variability and as BRET does not

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require an external light source it doesn’t suffer from photobleaching or sample autofluorescence and can reduce sample scattering making it more practical for measurement in

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complex and turbid media such as milk. Since BRET is also more compatible with turbid samples than colorimetric and fluorometric assays, we reasoned that it might be ideal for sensing plasmin

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activity in milk. Therefore, we set out to test the hypothesis that a BRET-based biosensor could deliver sufficient sensitivity to measure plasmin activity in raw milk within minutes, i.e.

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potentially fast enough for routine analysis of raw milk on receival at a processing plant. We designed a sensor for detecting plasmin with a similar overall architecture as BRET-based

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biosensors for thrombin and caspase-3 activities [16-18]. However, we added novel features designed to confer sensitivity and selectivity for plasmin. The plasmin biosensor comprises a peptide target sequence with cleavage sites from casein known to be specifically targeted by

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plasmin. The target sequence is flanked by the bioluminescence resonance energy transfer (BRET) transduction domains: green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus (Figure 1 (a)). Plasmin cleavage of the target decouples the BRET donor and acceptor, radically reducing the efficiency of Förster resonance

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energy transfer between them.

Figure 1

2. Methods 2.1. Construction of Sensors 5

DNA constructs encoding the BRET-plasmin sensor were synthesised by GenScript (USA). The constructs were cloned into a pRSET-CFP vector (BioLabs, Australia), using EcoRI and XhoI restriction sites, to make a pRSET-plasmin sensor vector encoding the plasmin sensor. Construction of analogous pRSET-thrombin sensor and pRSET-RLuc2 vectors have been described previously [18].

2.2. Expression and purification

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Proteins were expressed in Escherichia coli strain BL21(DE3) (Novagen). An overnight culture was grown from a single colony in lysogeny broth (LB) (10 g tryptone, 5 g yeast extract, 5 g NaCl

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(pH 7.4) containing 100 ug/mL ampicillin and 2% glucose) at 37oC, 200rpm. Expression was induced by inoculating 250 mL LB containing 100 µg/mL ampicillin to an Abs600 of 0.1 and

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incubating at 37oC and 200 rpm for 4.5 hours, followed by overnight incubation at 22oC (200 rpm). Cells were harvested 24 hours after inoculation, by centrifugation at 4,300 x g (4oC) for 15

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minutes and resuspended in equilibrium buffer (57.7 mM Na2HPO4, 42.3 mM NaH2PO4, 300 mM NaCl, pH 7.0). The cell suspension was passed through a homogenizer (Microfluidics M-100P

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(Newton, Massachusetts, USA)) at a pressure of ≈144,790 KPa to give a crude cell-free supernatant, which was clarified by centrifugation at 15,000 x g (4oC) for 15 minutes.

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Proteins were purified using cobalt affinity chromatography (TALON® Superflow Metal Affinity Resin (Takara Clontech, Australia)) according to the manufacturer's instructions. Following elution of the purified protein with 150 mM imidazole, the sample was dialyzed against plasmin cleavage buffer (50 mM Tris (pH 8), 10 mM NaCl and 25 mM lysine) using a cellulose dialysis membrane with nominal 12 kDa cutoff (Sigma). Aliquots of 500 µL of the purified protein were

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snap-frozen on dry ice and stored at -80oC. Protein concentrations were estimated by measuring Abs280 nm.

2.4. Protease assays

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Plasmin from human plasma (P1867,  2 units/mg protein), human thrombin (GE27-0846-01,  2000 NIH units/mg protein (1 NIH unit = 0.324 ± 0.073 µg), bovine plasmin ((10602370001,  2 units/mg protein), cathepsin B (C6282,  10 units/mg protein) and cathepsin D (C3138,  5 units/mg protein) from bovine spleen, pepsin (10108057001, ~ 2000 units/mg protein) and human matrix metalloprotease -7 (M4565, 5000 units/mg protein) were all purchased from Sigma-Aldrich.

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2.4.1. BRET assays

Protease assays were carried out in a final volume of 100 µL in 96-well plates. Purified sensor

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(10 nM) was incubated for 10 minutes at 28 oC in plasmin cleavage buffer (50 mM Tris-HCl, 50 mM NaCl, 25 mM lysine, pH 8) or thrombin cleavage buffer (50 mM Tris [pH 8.0], 100 mM NaCl, and

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1 mM ethylenediaminetetraacetic acid [EDTA]) with varying amounts of protease added. Plasmin assays were carried out with or without the addition of 50 µL of full fat UHT milk or skimmed UHT

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milk. The BRET2 signal was measured by adding 5 µL of 100 µM Coelenterazine 400a (Clz400a) (Cayman Chemicals) following the 10 minute incubation.

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Simultaneous dual emission BRET measurements were carried out with a POLARstar OPTIMA microplate reader (BMG LabTech, Australia) using a BRET2 emission filter set, comprising an RLuc2/Clz400a emission filter (410 nm, bandpass 80 nm) and a GFP2 emission filter (515 nm,

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bandpass 30 nm). The integration time was set to 0.5 second.

2.4.2. Colorimetric assays

Using the chromogenic substrate D-Val-Leu-Lys-4-nitroanilide dihydrochloride (Sigma Aldrich)

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plasmin assays were carried out by mixing 50 µL of full fat UHT milk with different concentrations of human or bovine plasmin in plasmin cleavage buffer and 45 µL of chromogenic substrate followed by incubating the mixture at 37 oC for 120 minutes. The absorbance was measured at 405 and 490 nm every 2 minutes using a plate reader (Spectra Max M2, Molecular Devices, USA).

2.5. Data analysis 7

2.5.1. BRET method data analysis. BRET2 ratios were calculated as the ratio of emissions at 515 nm and 410 nm [16-18].

𝐵𝑅𝐸𝑇 𝑟𝑎𝑡𝑖𝑜 =

𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑎𝑡 515 𝑛𝑚

(Equation 2.1.)

𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑎𝑡 410 𝑛𝑚

All data are reported as means ± standard deviations (SD) (n=3) unless otherwise stated. BRET ratios were normalised between 0 and 100 % using the normalisation function in Graphpad Prism

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7 for Windows.

Data were fitted with a Log [Agonist] vs normalised response – variable slope model and the half

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maximal effective concentration (EC50) value fitted by GraphPad prism using equation 2.2 [19].

(Equation 2.2.)

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2.5.2. Colorimetric assay data analysis.

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𝑌 = 100/(1 + 10^((𝐿𝑜𝑔𝐸𝐶50 − 𝑋) × 𝐻𝑖𝑙𝑙𝑠𝑙𝑜𝑝𝑒

The absorbance at 490 nm was subtracted from the absorbance at 405 nm and plotted versus

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time. The rate of change in absorbance between 0 and 120 minutes was calculated for each added plasmin concentration (Figure S2). To construct calibration curves the initial velocity (0 – 40 minutes) was plotted against plasmin concentration and fitted with a second order polynomial

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model [19](R2 > 0.99) using Graphpad Prism 7 for Windows (Figure S3).

2.5.3. Detection limits and statistical measurements. All measurements were carried out in triplicate and reported as mean ± standard deviation unless

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otherwise stated.

Detection limits were calculated as the blank signal (Yb)+ 3D, where D = the standard deviation of the blank signal [20]. 𝑌 = 𝑌𝑏 + 3𝜎𝐷

(Equation 2.3.)

Detection limit were converted to concentration values by interpolating x values using the calibration curves. 8

Two-tailed unpaired t-tests with Welch’s correction were performed using Graphpad Prism 7 for Windows. Statistical significance was defined as P ≤ 0.05.

3. Results and discussion

3.1. Sensor design

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Proteases preferentially hydrolyse peptide bonds of polypeptide substrates that have compatible amino acids preceding and/or following the cleavage site. The general nomenclature of protease

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substrate positions was formulated by Schechter and Berger [21] (Figure (1b)).

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A number of studies have documented the preferred recognition sequences for plasmin, based on its natural substrates as well as synthetic peptide substrates. Plasmin cleaves proteins on the

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carboxyl side of lysine and arginine residues (P1 =Lys or Arg). It has a strong preference for an aromatic residue at the P2 site, as demonstrated by its known physiological substrates:

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osteocalcin (P2= Tyr), vitronectin (P2=Tyr), PAR1 (P2 = Tyr) and factor Xa (P2 = Phe) [22]. Combinatorial studies have shown that preferred tetrapeptide recognition sequences for plasmin preceding the cleavage site (i.e. P4  P1) are encompassed by P4 =Lys /Nle/Val/Ile/Phe, P3=Xaa

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(non-specific amino acid), P2 = Tyr/Phe/Trp/Lys and P1 = Lys. The majority of the extended substrate specificity for plasmin resides in the P2 and P4 residues with a moderate preference for lysine at P4 [23]. In milk, plasmin hydrolyses κ, αs1-, αs2- and β-caseins [24-26]. RP-HPLC results indicated that plasmin preferentially cleaves lysine residues located in the centre of αs1casein. Principal cleavage sites in this region including Lys102-Lys103, Lys103-Tyr104, Lys105 – Val106

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and Lys124-Glu125 [26]. Based on these principles, we designed a novel plasmin-specific target sequence by incorporating multiple plasmin cleavage sites from αs1-casein into the linker (Figure 1 (b)) and maintaining the preferred P4P1 tetrapeptide sequence (Lys-Lys-Tyr-Lys) for extended plasmin specificity [23]. The sequence of the plasmin specific target peptide was LQGSKKYKVKEGSLQ, which we flanked with BRET donor and acceptor sequences (Figure 1 (a)). Commercially

available

synthetic

chromogenic

(D-valyl-L-leucyl-lysyl-4-nitroanilide)

or

fluorogenic substrates (N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amido-4-methyl coumarin) 9

for detection of plasmin activity appear to be designed around plasmin specificity for hydrolysing peptide bonds with lysine in the P1 position. However, they do not maintain the preferred P4P1 sequence for extended plasmin specificity.

3.2. Plasmin specificity Addition of excess (10 nM) human plasmin to the plasmin biosensor resulted in an 87 % reduction

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in the BRET ratio from 1.00 ± 0.01 to 0.13 ± 0.08 within ten minutes (Figure 2). There was no significant difference (P=0.05) between the final BRET ratios measured with 10 minute (0.13 ±

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0.08) or 20 minute (0.11 ± 0.04) incubation times (Figure 2). Incubations of 10 minutes were used for all subsequent plasmin assays.

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Figure 2

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Besides plasmin, several other proteases, including thrombin [27], cathepsin B [28]and cathepsin D [29] occur naturally in milk. Due to the fact that thrombin and plasmin are both enzymes of

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the blood-clotting system and serine proteases we investigated the effect of thrombin addition on the plasmin sensor as indicator of the specificity of the target peptide substrate for plasmin. No measurable change in BRET ratio (P<0.05) was observed over ten minutes in the presence of

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10 nM thrombin. The same quantity of thrombin completely cleaved an analogous thrombinspecific sensor in ten minutes, demonstrating that the thrombin was active (Figure 3). Figure 3

It was also shown that 10 nM plasmin did not cleave a thrombin specific sensor consisting of BRET

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components separated by a thrombin specific linker sequence (LQGSLVPRGSLQ) [18]. We concluded that the plasmin-induced change in the BRET ratio of the plasmin sensor is due to cleavage of the plasmin specific linker (LQGSKKYKVKEGSLQ) rather than direct proteolysis of the BRET donor or acceptor, which are identical in the plasmin and thrombin biosensors. This was confirmed by SDS-PAGE, which showed that the His-tagged plasmin sensor, a single 69.6 kDa band, is cleaved into two bands by incubation with 10 nM plasmin. The new bands, at 31.5 kDa and 35.9 kDa, correspond to the predicted masses of His-tagged GFP2 and RLuc2, respectively. 10

The single sensor band at 69.6 kDa remained intact following incubation of thrombin with the plasmin sensor or when plasmin was incubated with the thrombin sensor (Figure S1, supplementary data). To further investigate the specificity of the plasmin sensor we investigated the response of the plasmin sensor to different classes of proteases. We investigated the response of the plasmin sensor to the endogenous milk proteases such as the cysteine protease cathepsin B and the aspartyl protease cathepsin D. We also investigated the response to pepsin and a representative

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metalloprotease, matrix metalloprotease-7 (MMP-7). No measurable change in BRET ratio (P < 0.05) was observed with the addition of 10 nM cathepsin B, pepsin or MMP-7 (Figure 3).

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addition of 10 nM cathepsin D resulted in a 38 % reduction in the BRET ratio from 1.00 ± 0.03 to 0.62 ± 0.05. It has been reported that cathepsin D can hydrolyse caseins, including αs1-caseins.

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The amount of immunoreactive cathepsin D and procathepsin D in bovine milk was estimated to be 0.4 µg/L (8.9 nM)[29]. However, only a small fraction of this was shown to be active with the

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major form being the inactive proenzyme procathepsin D. This suggests that the levels of naturally occurring active cathepsin D present in bovine milk are at much lower concentrations

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than those tested here. However, even in excess concentrations plasmin is 8 x more active towards the plasmin sensor than cathepsin D (Figure 3) demonstrating the superior specificity of

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the sensor for plasmin.

3.3. Sensitivity of the plasmin biosensor We measured the sensitivity of the sensor to different amounts of human and bovine plasmin in

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plasmin cleavage buffer, 50 % skimmed milk or 50 % full fat UHT milk (Figure 4). In all cases, data could be fitted successfully by a “Log10[Plasmin] vs normalised response with variable slope” model with correlation coefficient R2 = 0.99. Figure 4 The half maximal effective concentration (EC50 ± standard error) was calculated to be 0.54 ± 0.02 nM in buffer, 5.26 ± 0.34 nM in 50 % (v/v) skimmed UHT milk and 5.87 ± 0.12 nM in 50 % (v/v) 11

full fat UHT milk with a human plasmin standard. Using a bovine plasmin standard resulted in an EC50 of 2.00 ± 0.15 in buffer and 5.97 ± 0.59 nM in 50 % (v/v) full fat UHT milk (Table 1). The limit of detection for human plasmin in buffer was 0.03 nM (0.002 µg/ml) and the response was linear up to 3.29 nM (0.28 µg/ml) (Table 1). The limits of detection for human plasmin in 50% skimmed milk were 0.09 nM (0.008 µg/ml) and 0.25 nM (0.02 µg/ml) in 50% full fat UHT milk. The increase in the limit of detection may be partly attributed to differences in the variability of the BRET signal measured in full fat milk compared to skimmed milk as the standard

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deviation of the blank increased from 0.66 % in skimmed milk to 1.02 % in full fat milk samples. The log10 [Plasmin]-BRET response curves for skimmed and full fat milk can be superimposed

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(Figure 4) and there was no statistically significant difference between the EC 50 values (P=0.12) measured in the presence of either full fat or skimmed milk. This demonstrates that up to 1.5 %

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fat in the assay does not interfere with the biosensor. Use of the BRET-based biosensor is therefore potentially much simpler than current standard methods. Until now, measuring

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plasmin activity has had to be done on skimmed milk [11] or has required a centrifugation step to separate the cream phase [8, 10] and reduce the turbidity of the sample. In an alternative

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method, Rauh et al. (2014) avoided the need to remove fat prior to colorimetric assay readout by using a reference wavelength. The ratiometric nature of the BRET measurements used here builds in an internal referencing mechanism for the assay, enabling direct measurement of

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plasmin in turbid fat-containing milk samples.

The limit of detection for bovine plasmin in buffer was 0.26 nM (0.02 µg/ml) and the response was linear up to 8.41 nM and the limit of detection was 0.86 nM in 50 % full fat UHT milk with a linear response up to 29.92 nM. The increase in the limit of detection with bovine milk compared

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to human plasmin in 50 % (v/v) milk may be partly attributed to differences in the variability of the BRET signal measured with bovine plasmin and human plasmin as the standard deviation of the blank increased from 1.02 % with human plasmin compared to 2.22 % with bovine plasmin. The respective detection limits of 0.25 nM and 0.86 nM for human and bovine plasmin in full fat milk (Table 1) are below the naturally occurring levels of plasmin previously reported in raw and processed milk. Using a fluorogenic substrate, these ranged from 1.64 – 8.59 nM (0.14 – 0.73 µg/ml) in pasteurised milk [10] and 1.76 - 3.76 nM (0.15 - 0.32 µg/ml) in raw bulk milk from [30]. 12

This highlights that plasmin is resistant to heat treatment and that the pasteurisation can result in higher plasmin levels in the processed milk potentially due to the activation of plasminogen (the inactive precursor of plasmin) during storage [6].

The BRET-based plasmin biosensor

described here, therefore has the potential to be used to directly measure naturally occurring plasmin levels in raw and UHT milk samples in a ten minute assay.

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Table 1. Detection limits and linear range for plasmin biosensor in plasmin cleavage buffer, 50 % skimmed milk (v/v in buffer) or 50 % full fat milk (v/v in buffer) spiked with a range of human and

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bovine plasmin concentrations and incubated for 10 minutes at 28 C.

LODb

Human plasmin

Linear rangec

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EC50a

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[Plasmin] ((nM (µg/ml))

0.54(0.05)

0.03 (0.002)

Skimmed milk

5.26 (0.44)

0.09 (0.008)

0.54 – 51.52 (0.05 – 4.38)

Full fat milk

5.87 (0.50)

0.25 (0.02)

0.78 – 44.15 (0.07 – 3.75)

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Buffer

0.09 – 3.29 (0.008 – 0.28)

Buffer

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Bovine plasmin

Full fat milk aEC 50

2.00 (0.17)

0.26 (0.02)

0.48 – 8.41 (0.04 – 0.71)

5.97 (0.51)

0.86 (0.08)

1.19 – 29.92 (0.10 – 2.54)

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half maximal effective concentration; bLOD, Limit of detection; cThe linear range defined as being between 10 % and 90 % bioluminescence resonance energy transfer respon

3.3.1. Comparison of plasmin sensor with a colorimetric assay To enable a direct comparison between the performance of the plasmin sensor and a colorimetric assay we measured the response to different amounts of human and bovine plasmin (Figure S2) using a commercial chromogenic substrate and plotted calibration graphs (Figure S3) tested in the same assay medium (50 % full fat UHT milk in plasmin cleavage buffer) used with the plasmin 13

sensor. The detection limits for human and bovine plasmin were 1.80 nM and 0.50 nM, respectively (Table 2) determined with the chromogenic substrate and a 40 minute assay time. A detection limit of 0.16 nM (0.014 µg/ml) was determined with the same chromogenic substrate [11] in diluted (10 ×) skimmed milk samples with an assay time of 30 minutes (Table 2). The plasmin sensor exhibited lower detection limits of 0.25 nM and 0.86 nM for human and bovine plasmin, respectively, in 50 % (v/v) in full fat milk compared to the colorimetric assay and achieved this a much shorter 10 minute assay time. The bovine plasmin showed higher activity

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towards the chromogenic substrate compared to human plasmin with measured bovine plasmin activity being 3.5 × higher than the corresponding human plasmin activity (Figure S3). The

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difference in human and bovine plasmin activity measured with the chromogenic substrate corresponds to the difference in Michaelis constant (Km) measured with a fluorogenic substrate

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(Boc-Val-Leu-Lys-MCA) which incorporates the same plasmin recognition site as the chromogenic substrate used here. The Km values estimated with the fluorogenic substrate were 2.5 × 10-4 M

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for bovine plasmin compared to 7.7 × 10-4 M for human plasmin [31].

3.3.2. Comparison with other reported methods for measuring plasmin The sensitivity of the plasmin biosensor we describe compares well with other plasmin assays

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reported in the literature (Table 2). Many of the existing plasmin assays require longer incubation times than the plasmin sensor assay (Table 2). Electrochemical and acoustic sensors have also been developed for plasmin activity measurement and have detection limits between 0.56 and 0.65 nM plasmin in buffer using assay

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times of 60 minutes [32-34]. The most sensitive plasmin assay reported in the literature was ELISA based and had a detection limit of 0.03 nM (0.005 µg/ml) for plasmin in buffer, which is the same as that determined for human plasmin with the plasmin sensor in buffer (Table 1). The ELISA required multiple incubation and wash steps, resulting in assay times greater than 3 hours [35]. Another obvious difference is that ELISA quantifies the mass of plasmin, whereas the plasmin biosensor described here quantifies activity.

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Table 2. Comparison of plasmin assay methods described quantitatively in the literature and herein*. Linear range

LODd

Time

Assay

source

(nM)

(nM)

(minutes)

medium

Human

0.09 – 3.29

0.03

10

Bovine

0.48 – 8.41

0.26

10

Human

0.78 – 44.16

0.25

10

Bovine

1.19 – 29.92

0.86

Human

NDc

Bovine Bovine

c

ND

ELISAb

Bovine

0.06 – 0.89

Electrochemical

Bovine

1-20

Acoustic

Human

aChromogenic

TB

*

50 % milk/TB

*

10

50 % milk/TB

*

1.80

40

50%milk/TB

*

0.50

40

50%milk/TB

0.16

15-30

10 % milk/TB

* [11]

0.03

180

PB-Tg

[35]

0.56

60

PBf

[32]

0.59

60

TB

[33]

Bovine

ND 1-20

0.65

30

PB

[34]

Bovine

ND

0.50

< 2

PB

[36]

ND

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c

-p

*

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Colorimetrica

Reference

TBe

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BRET assay*

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Plasmin

Assay method

substrate is H-D-valyl-L-leucyl-lysyl-4-nitroalinide; bELISA, Enzyme linked immunosorbent assay; cND, Not determined; dLOD, Limit of detection, eTB, Tris buffer solution; fPB, Phosphate buffer solutionPB-T, gPB + 0.005 % Tween.

Many of the plasmin assays reported in the literature used bovine plasmin as a calibration

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standard [11, 32, 34, 35]. It has been reported that bovine and human plasmin contain the same structure and functional domains and the major difference between the two is the ability of their respective plasminogen precursors to be activated to plasmin by the plasmin activator, streptokinase [37].

Human plasminogen is readily activated by streptokinase but bovine

plasminogen is rendered resistant to streptokinase activation.

This is attributed to the

substitution of Ile558 for Val561 at the activation site in bovine plasminogen. Although the chromogenic substrate showed different activities for human and bovine plasmin the log10 15

[Plasmin]-BRET response curves for human and bovine in full fat milk can be superimposed (Figure 4b) and we observed no statistically significant difference between the EC50 values (P=0.89) measured with human and bovine plasmin. This demonstrates that it is reasonable to use human plasmin as a standard for determining bovine plasmin activity in milk samples using the plasmin sensor.

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3.4. Reproducibility of the plasmin sensor Initial assessment of biosensor reproducibility was determined from repeated measurements in

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buffer. Relative Standard Deviation (RSD) of the sensor response in buffer (0 % UHT milk, Figure S2, supplementary material) to 1 nM bovine plasmin was 16.9 % (n=3) compared to a RSD of 3.1

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% (n=3) measured with 1 nM human plasmin. This is consistent with the higher detection limits determined with the plasmin sensor (Section 3.3) for a bovine plasmin standard compared to a

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human plasmin standard which was attributed to increased standard deviation of the bovine plasmin blank compared to that of human plasmin. The improvement in sensor reproducibility

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with human plasmin compared to bovine plasmin could arise from the difference in the commercial plasmin preparations. Human plasmin is provided as a lyophilized powder which is reconstituted in plasmin cleavage buffer, whereas, bovine plasmin is provided as an insoluble

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suspension in 3.2 M ammonium sulfate solution at concentrations. The improvement seen in reproducibility with human plasmin compared to bovine plasmin makes it the preferred choice for use as a standard for measuring plasmin activity. As casein levels in milk may vary between samples we measured the sensor response in ten

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individual full fat UHT samples spiked with 5 nM of human plasmin to assess the reproducibility of the method (Figure S2, supplementary material). The RSD for the sensor response in UHT milk samples without the addition of any human plasmin was determined to be 2.3 % (n= 10, x̄ = 3.98). Following incubation with the same UHT samples spiked with human plasmin the RSD was determined to be 6.71 % (n=10, x̄ = 2.68). The change in BRET ratio in response to human plasmin addition to milk samples was 32.62 ± 3.90 % (n=10) with a calculated RSD of 12.0 % (n=10).

16

3.5. Plasmin activity in milk samples To characterise the sensitivity of the plasmin biosensor to possible interferents in milk we investigated the effect of the proportion of milk in the assay. We used the sensor to measure the proteolytic activity of human and bovine plasmin in the presence of an increasing proportion of full fat UHT milk (Figure 5). Measured proteolytic activity decreased as the proportion of UHT

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milk in the assay was ramped up. For example, 20 % (v/v) milk reduced the activity of human plasmin by 88 % and bovine plasmin by 61 %, relative to buffer control and 50 % (v/v) milk

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reduced activity of human plasmin by 94 % and bovine plasmin by 79 % (Figure 5 a). The proteolytic activity of 5 nM plasmin was less inhibited by increasing the proportion of UHT milk

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than 1 nM plasmin (Figure 5 b). For example, 50 % (v/v) milk reduced the activity of 5 nM human plasmin by 58 % and 1 nM human plasmin by 94 % and 5 nM bovine plasmin by 63 % compared

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to 79 %, relative to the buffer control. The reduction in plasmin activity relative to the buffer control was not significantly different (P=0.225) for 5 nM human and bovine plasmin in 50 % (v/v)

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milk (Figure 5b). This is consistent with no statistical difference being observed between the EC50 values (P=0.89) for human and bovine plasmin in 50 % (v/v) milk (Table 1). The suppression of plasmin activity in milk is attributed to inhibitory effects of caseins and whey proteins on plasmin

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activity. This is supported by the observation that casein competitively inhibited plasmin activity measured using a chromogenic substrate [14]. The greater inhibition of human plasmin activity compared to bovine plasmin by UHT milk is consistent with the 10- and 3- fold shifts seen in the log10 [Plasmin]-BRET response curves in full fat UHT milk compared to buffer (Figure 4). The greater inhibition of human plasmin activity in UHT milk compared to bovine plasmin activity

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could be due to the difference in their respective sequences and structures. Following conversion of bovine and human plasminogen to plasmin via autolytic cleavage, bovine plasmin undergoes an additional autolytic cleavage at Arg342-Met343 between kringle domains 3 and 4 to produce midi plasmin [38]. The Arg342 – Met343 bond that is cleaved in bovine plasmin corresponds to Glu342-Leu343 bond in human plasmin, which is not susceptible to plasmin digestion.

Midi

plasmin is predominantly found in the serum (non-casein) fraction of milk demonstrating that

17

bovine midi-plasmin is more weakly associated with casein than full-length human plasmin, presumably because it has lower lysine-binding capability than human plasmin [39]. Figure 5 The inhibitory effects of milk on measured plasmin activity reported here are larger than those reported by Rauh et al. (2014) and Saint-Denis et al. (2001) using colorimetric and fluorometric substrates, respectively. Rauh et al. (2014) reported a 52 % loss in activity in undiluted full fat

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UHT milk and a 29 % drop in 20 % (v/v) milk. Saint-Denis et al. (2001), using a fluorometric substrate, reported a 30 – 40 % loss of activity in 25 % (v/v) milk. Rauh et al. (2014) and Saint-

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Denis et al. (2001) used substrate concentrations of 1-2 mM in the reaction mixture to compensate for the casein present in the milk samples being analysed, whereas, we used a sensor

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concentration of 10 nM which could account for the larger inhibitory effects of UHT milk on plasmin activity when measured with the plasmin sensor. However, we were able to achieve

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sufficient sensitivity to measure naturally occurring levels of plasmin in raw and UHT milk samples using a much lower substrate concentration (100,000 -200,000 x) than previously reported for

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Conclusions

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colorimetric and fluorescent substrate-based assays (Table 2).

We designed and characterised a plasmin specific BRET-based biosensor that has sufficient sensitivity and specificity to enable direct measurement of naturally occurring bovine plasmin levels in milk samples. We tested the plasmin sensor using human and bovine plasmin spiked into bovine milk. The plasmin biosensor was more sensitive than a chromogenic substrate when

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tested in 50 % full fat milk solutions and only required a 10 minute assay time compared to 40 minutes with the colorimetric assay. The sensitivity of the biosensor was similar using human or bovine plasmin in UHT milk, demonstrating that it is feasible to use a human standard to measure bovine plasmin in milk. The assay does not require wash or multiple incubation steps. Additional benefits including speed, convenience and sensitivity could be realised by deploying the sensor in a flow-format using a microfluidic device [40, 41]. This would also enable continuous monitoring offering a promising approach for in-line detection at a processing plant. 18

In milk, plasmin associates with casein micelles and several pre-treatment protocols have been developed to dissociate plasmin from caseins to enable accurate determination of total plasmin levels in milk samples. Whilst outside the scope of this study, we have optimised a pre-treatment protocol and demonstrated the use of the plasmin biosensor described here, to accurately measure plasmin activity in raw milk samples, which is the subject of a follow up paper.

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Acknowledgments The authors would like to acknowledge Dr Mustafa Musameh and Dr Felix Weihs for their critical

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review of the manuscript.

19

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of plasminogen and plasmin from bovine milk, International Dairy Journal, 5(1995) 577-92. [40] N.C.H. Le, M. Gel, Y. Zhu, J. Wang, H. Dacres, A. Anderson, et al., Sub-nanomolar detection of thrombin activity on a microfluidic chip, Biomicrofluidics, 8(2014). [41] N.C.H. Le, M. Gel, Y. Zhu, H. Dacres, A. Anderson, S.C. Trowell, Real-time, continuous detection of maltose using bioluminescence resonance energy transfer (BRET) on a microfluidic system, Biosensors & Bioelectronics, 62(2014) 177-81.

22

Biography Dr Helen Dacres is Team Leader of the Biosensors team at CSIRO in Australia and leads research in biosensing systems for food and health diagnostics and biosecurity surveillance. She received a PhD (2004) and MSc (2000) in Instrumentation and Analytical Chemistry from UMIST (Manchester, UK) developing optical chemical sensors to detect biologically and environmentally relevant gases including nitric oxide. She joined CSIRO as a postdoctoral fellow in 2005 to work on the development of biosensors using biological odorant receptors for detecting volatile compounds. Since joining CSIRO in 2005 Helen has pioneered the use of Bioluminescence Resonance Energy Transfer (BRET) in the development of range of different classes of CYBERNOSE®/CYBERTONGUE® biosensors which form the basis of a ‘tool-kit’ of biophotonic sensors capable of detecting a wide-range of proteins and chemicals. Helen has over 60 publications and is inventor on 8 patent families.

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Dr Jian Wang completed a BSc degree (Biology, 1982) at Hebei Normal University in China and a PhD (Biochemistry and Cell Biology, 1993) at The Australian National University. He made use of molecular biology, cell biology and biochemistry tools to study plant, human and animal health and development. Following two post-doctoral fellowships (CSIRO, 1993-1995 and RSBS, ANU, 1995-1997), Jian moved to John Curtin School of Medical Research as a Senior Research Officer for five years, where he carried out cancer research in collaboration with The Canberra Hospital. In 2002, Jian joined Professor Richard Williamson’s Plant Cell Wall Group as a Research Fellow (2002-2008) to study cellulose synthesis. Jian has been a Senior Technical Officer studying bio-sensory science in Biosensor team at CSIRO, Australia since 2008.

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Dr Alisha Anderson is the Group Leader of the Innovative Bioproducts group in CSIRO Health & Biosecurity. Alisha received her PhD from Monash University in 2005. She has a broad background in genetics and molecular biology. Her current research is focused on understanding the molecular basis of biological chemosensory systems and drawing on this understanding for new sensing technologies. Alisha leads the Innovative Bioproducts group who have developed the CYBERNOSE®/CYBERTONGUE® technology for rapid and sensitive chemical sensing across multiple industries including Defence, Water Utilities, Food & Beverage, and Health.

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Dr Stephen Trowell has held the position of CSIRO Honorary Fellow since 2018 and is founder of PPB technology. Stephen obtained his first degree, in biochemistry, from Cambridge University and a PhD in biochemical neuroscience from the Australian National University in 1987. He joined CSIRO in 1989 on an ARC National Research Fellowship. Stephen led the CSIRO team that developed and commercialised The LepTon™ Test Kit, an immunodiagnostic kit used to manage insecticide resistance in the cotton industry. Stephen developed CSIRO's insect extract library for drug discovery and invented the CYBERNOSE® and CYBERTONGUE® technology platforms. Stephen has well over 100 publications and is inventor on 14 patent families. He continues to serve on the Editorial Board of Bioinspiration and Biomimetics and in 2013 received a Newton-Turner Award. Stephen was elected a Fellow of the Royal Society of NSW in 2018.

23

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Figure Legends.

Figure 1 (a). Cartoon of the BRET based biosensor for measuring plasmin activity. Plasmin cleavage of the target sequence promotes dissociation of the bioluminescence resonance energy transfer (BRET) donor, Renilla luciferase (Rluc2) and acceptor, green fluorescent

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protein (GFP2). CLZ400a = Coelenterazine 400a. (b) i. Nomenclature of protease substrate specificity. The substrate is cleaved between amino acids P1 and P1’ [21]. Amino acid

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residues are numbered outwards from the cleavage site, ii. Design of the plasmin-specific

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substrate peptide (P4 –P4’ amino acids) incorporated into the plasmin biosensor.

24

Figure 2. Time dependency of plasmin action on the plasmin biosensor. Normalised bioluminescence resonance energy transfer (BRET) ratios (normalised by BRET ratio at time = 0) following addition of 10 nM human plasmin to 10 nM plasmin sensor for different times (0

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– 20 minutes), (Mean ± SD, n=3).

Figure 3. Specificity of plasmin biosensor. Normalised bioluminescence resonance energy

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transfer (BRET) ratio (normalised against the “no protease” BRET ratios (grey bars)) for the BRET-based plasmin and thrombin biosensors [18]. Incubations were with 10 nM plasmin, 10 nM thrombin, 10 nM cathepsin B, 10 nM cathepsin D, 10 nM pepsin, 10 nM matrix

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metalloprotease-7 (MMP-7) or no added protease, for 10 minutes at 28 C (mean ± SD, n=3),

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* P ≤ 0.05.

25

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Figure 4. Log10 concentration response curves for the plasmin biosensor in buffer, with and without addition of milk. Bioluminescence resonance energy transfer (BRET) response (mean ± SD, n=3) of the plasmin sensor after incubation (10 minutes at 28 C) with various concentrations of human plasmin in buffer, skimmed UHT milk (50 % in buffer (v/v)) and full fat UHT milk (50 % in buffer (v/v)). 26

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Figure 5. Effect of varying the proportion of milk on human and bovine plasmin activity measured with the plasmin biosensor in plasmin cleavage buffer. Relative activity (mean ± SD, n=3) of (a) 1 nM and (b) 5 nM of human and bovine plasmin in full fat UHT milk diluted in

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plasmin cleavage buffer compared to activity in plasmin cleavage buffer only (100 %).

27