Biochip point-of-care device for sepsis diagnostics

Sensors and Actuators B 192 (2014) 205–215

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Biochip point-of-care device for sepsis diagnostics Manuel Kemmler a,∗ , Ursula Sauer c , Erwin Schleicher b , Claudia Preininger c , Albrecht Brandenburg a a b c

Fraunhofer Institute of Physical Measurement Techniques, Freiburg, Germany University of Tuebingen, Department of Internal Medicine, Tuebingen, Germany AIT Austrian Institute of Technology, Tulln, Austria

a r t i c l e

i n f o

Article history: Received 22 May 2013 Received in revised form 24 September 2013 Accepted 1 October 2013 Available online 24 October 2013 Keywords: Point-of-care Biochip reader Fluorescence immunoassay Sepsis Inflammation Optical sensor

a b s t r a c t We report about a point-of-care device for the diagnosis of sepsis. For this a useful set of diagnostic parameters must be analyzed in parallel, to combine the diagnostic benefit of each parameter. By this the severity of sepsis can be diagnosed, predictive indicators can be gathered and it is possible to differentiate between viral and bacterial sepsis being all essential information for septic patients. Therefore the protein biomarkers C-reactive protein (CRP), interleukin 6 (IL-6), procalcitonin (PCT) and neopterin (NPT) are integrated in a multiparameter on-chip immunofluorescence assay. Furthermore important criteria for point-of-care testing (POCT) such as low testing time, small sample volume, compactness, cost efficiency, accurate fluid handling, automated operation, sensitive detection and good reproducibility are closely examined. The measurement system is based on Total Internal Reflection Fluorescence (TIRF) by use of special biochips. The system reads out microarray based multiparameter immunofluorescence assays. To characterize the device several standard curves were generated by three-fold replicates in a serum model with 4% human serum albumin and human plasma containing spiked standard analytes. PCT and IL-6 are carried out in a sandwich assay format whereas NPT and CRP are processed in a binding inhibition format. Both assay types are processed in parallel by the fluidic unit. The assay time for a single multiparameter assay is 25 min. An accurate execution of the two assays formats in parallel needs several fluid handling steps like dilution, mixing, metering, separating, pre-incubating and incubating to be carried out. The protein chip is processed fully automated using 10–75 ␮l human plasma or serum sample. An additional important demand for POCT is the accuracy and precision of the measurements. This paper shows a method how imprecision and inaccuracy can be drastically reduced by using on-chip reference assay signals to reference the sepsis parameter signals. The on-chip reference assay runs parallel to the multiparameter assay. The method is analyzed with standard curves using human plasma matrix. To investigate the performance of a new a point-of-care device, it is necessary to run the assays with clinical specimen of healthy and pathological patients. For this the IL-6 levels were first investigated by established clinical systems in two different hospitals and then compared with the new platform. It was possible to resolve the clinical relevant IL-6 levels and a required LOD in the lower pg/ml range was achieved. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Sepsis is one of the most common causes of death in intensive care units [1,2]. In severe cases of sepsis, a fast diagnostic result is essential due to a high mortality rate. By point-of-care testing (POCT) the therapeutic-turn-around-time is reduced, since the patient sample has not to be sent to a clinical laboratory [3]. Sepsis is always caused by a local infection; therefore it can be the result of every infection if the immune system is not able to

∗ Corresponding author. Tel.: +49 761 8857 742; fax: +49 761 8857 224. E-mail address: [email protected] (M. Kemmler). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.10.003

isolate the infectious origin. When sepsis is arising bacteria or viruses release toxic agents causing inflammation in all organs within several hours only; acute organ failure is the consequence. Finally the systemic immune response turns against the patient himself. At this point there is no chance of survival without intensive care. Even if the patient gets immediate intensive care with antibiotic treatment, the chance of survival is only 20–40%. Therefore an early diagnosis of sepsis is essential. A proper device for POCT should have a low testing time. Additionally compactness, automated processing and cost efficiency are important points for the decentralized use of the system. Furthermore small sample volumes, quantitative analyte detection and parallel multi-analyte detection are critical needs for clinical

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diagnosis of sepsis. At the moment there is no device at the market fulfilling all mentioned criteria to diagnose sepsis [3]. For the presented platform microarrays are used with fluorescence immunoassays as detection method being properly used to fulfil these points. Accuracy and imprecision are reported to be difficultly observed with this tool. However there are regulations concerning the issue of accurate measurements like the FDA “Guidance for Industry on Bioanalytical Method Validation” [4]. Tan et al. [5] reported that the results of commercially available biochip platforms for DNA tests are varying. The variances are caused by the platform, the processing and the analytical steps. In Ref. [6–8] similar restrictions are described. The quantification of genes and gene expression data, especially of low abundant ones, is not accurately achieved by the biochip technology. To solve this problem an improvement of chip preparation, the platforms and test conditions are stated as well as the use of proper standard protocols and references. Not only DNA based methods are limited, in [9] also the quantification with protein array is said to be difficult. There are few providers for standardization or calibration available like Fullmoon Biosystems, Clondiag Chip Technology, Fuji Photo Film and Axon Instruments. These standards are necessary to obtain long-term stability. The claimed points are similar to those before: standardization of instruments, protocols, tests and the establishment of proper reference materials. Nevertheless all of the cited publications attribute at high potential to the microarray technology if these limitations are hurdled. For the presented platform it was not possible to use the named standard chips to standardize the system since these formats were not suitable. 2. Experimental 2.1. Instrumentation The developed system is capable to measure four sepsis relevant markers within human matrices by use of protein microarrays. It comprises an optical detection unit and a fluidic unit being controlled by software including the array analysis. 2.2. Optical detection The system has a high sensitive read out via Total Internal Reflection Fluorescence (TIRF, [10–13]) generated in a special planar waveguide chip. The fluorophores get excited in the evanescent field at 638 nm with a temperature stabilized semiconductor laser. The system works with a cost efficient 8-bit fire wire CCD camera. This improves the portability, compactness and system price being important for use as a POC system. As seen in Fig. 1 tandem objective depicts the whole micro array on the CCD camera. The CCD camera is non-cooled and provides fire wire connection and 8-bit resolution optimized for near-infrared sensing of CY5, DY647 or similar fluorophores. To increase the dynamic range for signal detection the integration time of the camera is varied. By this several array images can be taken increasing the absolute dynamic range considerably. In the parallel beam path (see Fig. 1) the interference filter separates the excitation wavelength from emitted fluorescence. 2.3. Liquid handling unit The liquid handling unit is capable to process two assay formats fast, automated and parallel: a binding inhibition and sandwich assay format. Please see Fig. 4 in Section 2.6 for the description of the assay formats. In this section the integrated fluidic process is

Fig. 1. Schematics: read out. The fluorescence is excited at 638 nm by a laser coupled into a special planar waveguide biochip exciting the fluorescence by Total Internal Reflection Fluorescence (TIRF). The microarray is detected by a CCD camera via a tandem objective filtering the fluorescent light.

described in detail. Fluidic components, signal analysis and a more detailed description of the read out technique are reported in [11]. The automated liquid handling unit of the system has the following flow schema: The fluidic system is capable of performing multiparameter immunofluorescence assays for the clinical parameters CRP, IL-6, NPT and PCT. The system features the combination of conventional 2-step sandwich assay with a binding inhibition assay. CRP and NPT run in a binding inhibition format, whereas IL-6 and PCT run in a sandwich assay format. The procedure includes first diluting the sample with CRP and NPT antibodies by factor 10, and secondly incubating this mixture together with PCT and IL-6 antigens on the chip. In the following step fluorescent antibodies for IL-6 and PCT are added to complete the binding reaction and read-out the result on the micro array chip. Control of the fluidic operations is performed fully automated by the system software in a scripting process. The fluidic system is presented schematically in Fig. 2. The fluidic handlings protocol is performed as follows: Before starting the assay the whole system is flushed with system buffer (phosphate buffered saline, PBS). The probe and sample ports are not filled completely with PBS, therefore an air plug is left in the tubing. The system requests the user to hold the sample to the open end. The left air plug in the tubing separates the sample from the system buffer, when 10 ␮L of the sample is sucked into the tubing. The same steps are repeated at another port for the probe, whereas 90 ␮l are sucked in. The reagents get removed from the tubing and air is sucked in behind the fluids. Both fluid bulks are now located in front of the distributor valve. The syringe pump sucks the two fluids by alternating the ports of the distributor valve. This is done very precisely to merge the two separating air plugs to one air plug. After this merging the port is seven times repeatedly switched. Thereby the two fluids are mixed by factor 10 by the use of multilamination [14]. The sample is divided into three bulks and is mixed with four surrounding probe bulks. The result is a mixture with a total volume of 100 ␮l surrounded by air plugs. By pumping these 100 ␮l for- and backwards the parabolic velocity profile is inducing improved mixing conditions. A following idle period of 30 s is implemented. The distributor valve switches to the flow cell and the mixture is pushed in. Together with the biochip the flow cell builds a reaction chamber (please see Fig. 1). The separating air plugs are removed by the bubble trap being located directly in front of the flow cell. Then the sample incubates in a stop-flow-process for ten minutes in total. The flow cell has a height of 30 ␮m and therefore a very short diffusion length providing a fast incubation time. After 120 s

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Fig. 2. Liquid handling unit. A syringe pump is used in combination with a distributor valve and a flow cell as a liquid handling unit. Several containers for sample, probe, buffers and waste are providing the reagents needed for the assay. The unit is capable to process a complex series of liquid handling steps to reproduce a fluorescence immunoassay. This includes the features diluting, mixing, metering, separating by air plugs, air plug removal via a bubble trap, pre-incubating, incubating and washing. The microarray is located in a reaction chamber with 30 ␮m height build by the flow cell.

incubation time, 10 ␮L of the sample get pushed forward. This step is repeated five times. In the washing process 250 ␮L washing buffer and two times 250 ␮L PBS are pumped through the flow cell. Then the result measurement of the binding inhibition assay is done. Afterwards the system requests the user to keep the detection antibodies ready for the sandwich assay which is then pumped through the flow cell as well. Then a second on chip incubation process with the probe is performed. After 80 seconds incubation time, 10 ␮L of the probe get pushed forward. This step is repeated two times and after a second washing step the result measurement for the sandwich assay is performed. In the following the process for diluting and mixing will be described in detail. The syringe pump generates a pressure driven flow whereas the distributor valve generates multilaminated fluid bulks [14] consisting of sample and probe, please see Fig. 3. Due to the tubing dimension and the used velocity laminar flow conditions are present. Therefore the profile of the z direction of velocity vz (r)

in the tubing as a function of the radius r is generated following a parabolic function

vz (r) ∝ (R0 2 − r 2 ) R0 represents the inner radius of the tubing; therefore velocity vz at tube boundary is zero whereas in the centre the maximum velocity is present. Please see scheme in Fig. 3. The generated fluid bulks get mixed by the pressure induced flow profile and Taylor dispersion [15]. The increased surface area of the fluid bulks boundary layers linked with the parabolic velocity profile creates mixing conditions which do not only depend on diffusion. By changing the flow direction for several times this effect is enhanced. The surrounding air plugs avoid the sample– probe mixture is not diluted with system buffer. This assures high analyte concentration on the biochip and allows lower detection limits. By changing the volumes of sample and probe different dilution factors can be easily applied. For the following experiment a dilution factor of ten is chosen. Therefore the fluid bulks of the probe have a nine-fold higher volume then the sample fluid bulks. By these proper mixing conditions presented analytes are diluted and pre-incubated within the tubing. To summarize the described processes generate a mixture with an adjustable dilution factor. Mixing and pre-incubating are carried out being essential to perform binding inhibition or competitive assay formats. The whole process takes five minutes and is done in simple tubing. 2.4. Reagents

Fig. 3. Schematics: multilamination. Multilamination of sample (white) and probe (black) by pressure induced flow in combination with an active valve (grey) in a tubing with the radius R0 .

10× Phosphate buffered saline (PBS) (pH 7.2) was ordered from Invitrogen, Karlsruhe, Germany and diluted to a 1× concentration. PBS was free of CaCl2 and MgCl2 . Dilution buffer for detection antibodies contains 1:5 LowCross buffer from Candour, Weissensberg, Germany.

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Fig. 4. Assay formats. Sandwich assay: antibodies are spotted on the chip (black Y) and bind covalently to the epoxy chemistry. In a first step the antigens (grey rectangle) of the sample incubate with the spotted antibodies. In a second step the fluorescent labelled antibodies (grey Y with pentagon) incubate with the antigens. The dose–response curve shows high signals at high analyte concentrations. This assay format is used for IL-6 and PCT. Binding inhibition assay: Antigens are spotted on the chip (black rectangle) and bind covalently to the epoxy chemistry. In a pre-incubation step the fluorescent labelled antibodies (grey Y with pentagon) react with the antigens of the sample (grey rectangle) in solution. In the next step the non-bound labelled antibodies incubate on chip. The dose–response curve shows low signals at high analyte concentrations. This assay format is used for CRP and NPT.

Tween 20, human serum albumin and sodium deoxycholate was purchased from Sigma Aldrich, Taufkirchen, Germany. Blocking, washing and dilution solutions consist of 0.1% Tween 20 in 1× PBS (PBS-Tween). Spotting buffer was 0.001% sodium deoxycholate in 1× PBS. Monoclonal CRP antibody was purchased from EXBIO Prague, Vestec, Czech Republic (spotted: antigen, detection: antibody clone5) and CRP was obtained from Biodesign, Saco, USA. IL-6 antigens and antibodies (spotted clone: MQ2-13A5, detection clone: MQ2-39C3) were from eBioscience. PCT was purchased from Prospec-Tany Technogene Ltd., Rehovot, Israel. PCT monoclonal antibodies (PROC1 3G3 and PROC4 6B2 were produced and provided by Helmholtz Centre Munich–German Research Centre for Environmental Health (GmbH), Germany [16]. NPT monoclonal detection antibody and spotting bovine serum albumin conjugate were produced and provided by Veterinary Research Institute, Czech Republic. [17] Labelling of all monoclonal detection antibodies with fluorescent dye DY 647 was carried out by EXBIO.

2.5. Chip preparation The planar waveguide biochips are coated with SU-8 based ARChip Epoxy chemistry [18,19] via dipcoating. HSA test was spotted with the non-contact spotter TopSpot (Biofluidix) and human plasma test printed using the BioOdyssey Calligrapher Miniarrayer (Bio-Rad) contact spotter. Chip-to-chip corrections test and the clinical trials were spotted with the Qarray mini spotter (Genetix). 0.2 mg/ml of each CRP antigens, 0.25 mg/ml IL-6 and 0.5 mg/ml PCT antibodies were printed in 1× PBS (pH 7.2)/0.001% sodium deoxycholate and in several replicates onto the chip for the HSA test. 0.2 mg/ml of each NPT spotting conjugate, IL-6 and PCT antibodies were printed in 1× PBS (pH 7.2)/0.001% sodium deoxycholate and in several replicates onto the chip for the human plasma test. Other tests were spotted with 0.25 mg/ml NPT spotting conjugate, 0.5 mg/ml IL-6 antibodies and 0.085 mg/ml rabbit IgG as reference spots N(ref) were printed in 1× PBS (pH 7.2)/0.001% sodium deoxycholate and in several replicates onto the chip for the plasma test. After arraying the chips were stored at 4 ◦ C until use.

2.6. Assays The system processes two assay types in parallel; the binding inhibition assay for the parameters NPT and CRP and the sandwich assay for the parameters IL-6 and PCT. The fluidic processing is described in Section 2.3. In Fig. 4 the assays types are outlined. In the following the experimental protocols are described: Biochips are blocked for 30 min in PBS 0.1% Tween 20 on a thermal pack directly before usage. For the assays mixes containing the analyte concentrations were made. First dilutions are made by use of one master mix consisting of the highest concentration of each analyte. Then this master mix is diluted e.g. 1:10, 1:100 and 1:1000. For the 4% HSA test the dilution buffer is PBS supplemented with 4% (w/w) HSA which is also used as the zero sample (0 ng/ml biomarker). For measurements in plasma antigens were spiked into plasma of healthy human donors. Since physiological values for these two parameters are below the system sensitivity no offset is present. Plasma and serum samples containing anticoagulants were obtained from collected blood samples by centrifugation at 3000 × g for 10 min at 4 ◦ C. Participants signed a written informed consent being approved by the Ethical Committee of the University Hospital of Tuebingen and Munich. Protein biomarkers were analyzed by the routine autoanalysers as described in the text. The respective imprecision also is noted. A second master mix is called diluting mix, since this mix is used to dilute the sample. It is containing the detection antibodies for the binding inhibitions assays and/or the reference reaction antibodies. For the 4% HSA test the diluting mix consists of detection antibodies for the CRP binding inhibition assay: 2 ␮g/ml. For the plasma test the diluting mix consists of 100 ng/ml DY 647 labelled NPT antibody. For the referencing test 100 ng/ml DY 647 labelled NPT antibodies and 20 ng/ml Alexa 647 labelled anti-rabbit IgG were used. The clinical trial diluting mix contained only 20 ng/ml Alexa 647 labelled anti-rabbit IgG. The third master mix contains all detection antibodies for the sandwich assay: DY 647 labelled PCT and IL-6 antibodies with a concentration of 1.11 ␮g/ml for the plasma tests and 2 ␮g/ml for IL-6 for all other tests. These master mixes are used for all measurements. For the experiments the dilution factor of the samples was varied by factor 10, 4 and 0.75. In the experiments were sample is diluted by factor 10, 10 ␮L of sample and 90 ␮L of the diluting mix was used, for a factor 4 dilution 25 ␮L of sample and 75 ␮L of the diluting mix and for a factor 0.75 dilution 75 ␮L of sample and 25 ␮L of the diluting mix are used.

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Fig. 5. Spot analysing. By pattern matching the software image processing builds a square around the image of the spots. The useful signal of the spot is calculated by subtracting the background signal (grey edges) from the spot signal (black circle).

Finally 100 ␮L of the third master mix containing the sandwich assay probe were used for each chip. 2.7. Data analysis A robust and reproducible method for the analysis of the fluorescence images of the spots is needed. The evaluation of the raw data is explained by using Fig. 5. The useful spot signal s is the subtraction of the spot signal and its local background. In the grey edges the median of the background pixels p˜ b is build. This value is subtracted from each pixel pi within the black line by resulting in the background corrected signal sb : sb =

n 

(pi − p˜ b )

i=0

To extend the dynamic range of the 8-bit camera the exposure time is varied from 0.01 to 60 s. The grey values of the pixels were investigated to have liner correlation with the exposure time. Because of this the array pictures are taken for various integration times, for each spot the exposure time with the maximum signal to noise ratios determined by the software by using the equation. The signal of the spots sb is normalized by building the quotient of a constant reference time tref and the actual exposure time texp forming the background and exposure time corrected signal value S, used for all subsequent calculations: S=

sb · tref tint

In the following chapters standard curves are processed to characterize the system performance. For the calculations the FDA guidance for bioanalytical method validation [4] will be respected. As described in [20] and recommended the FDA guidance the 4parameter logistic fit model is suitable to fit the dose response curve of immunoassays. According to this, the following relation between the analyte concentration cfit and the signal Sfit is given: Sfit (cfit ) =

A−D 1 + (cfit /C)

B

+D

The parameter A corresponds to the zero concentration Sfit (0) response; B is the slope factor, C the inflection point and D the infinite concentration response. The limit of detection LOD and the lower limit of quantification LLOQ are calculated by the signal Sfit (0). The parameter truec determines the true value of the analytes concentration generated by pipetting the standard solution. For the data analysis the following values are calculated:

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Fig. 6. Scheme reference spots. The signals of the upper and lower reference-spots are used a correct the signals of the sepsis-parameter-spots by a quotient based function.

2.8. Referencing For the improvement of spot-to-spot and chip-to-chip variances a direct test is implemented on chip. A spotting reference antibody is spotted with a concentration of 0.085 mg/ml on the chip. The reference antibody reacts with a detection reference antibody in a direct immunoassay format. The detection reference antibody is mixed with the sample with a concentration of 20 ng/ml. The spotting reference antibody is spotted close-by the spotting antibody/antigens of the sepsis parameter detection. This means, the reaction of the immunoassay for the sepsis parameters is parallel to the reference reaction. Since the used concentrations of the reference antibodies are constant during all measurements a theoretically error-free system should show zero percent deviation for the signal of reference spots. Indeed numerous errors are represented in the signal of these reference spots. In the following the calculation to use this signal for the correction of errors will be described: As it is seen in Fig. 6 the reference spots are positioned in two rows above and below the sepsis parameter spots. Denoting the signals S as a function of row m and column n of the array, where S(n,m − 1) and S(n,m + 1) are reference spots, while S(n,m) is representing a sepsis parameter. The signal value after referencing Sdot-ref is calculated as follows: Sdot-ref =

S(n,m) ((S(n,m−1) + S(n,m+1) )/2)

By referencing errors from e.g. in coupling errors, damping effects in the waveguide and errors from incubation or other fluidic operations are corrected. By building the median throughout the column n of S(n,m) one value of each sepsis parameter per chip is calculated. This is performed for the corrected data Sdot-ref and the original (not corrected) data Sorg . Since the improvement of this referencing method will be investigated the calculations from Table 1 will also be performed for the corrected Sdot-ref and the original data Sorg . 3. Results and discussion 3.1. Standard curves in 4% human serum albumin To characterize the system, analyte standards are spiked in a human matrix to determine the limit of detection (LOD), inaccuracy and imprecision following the guidance for industry for bioanalytical method validation [4]. The matrix is PBS containing 4% human serum albumin (HSA) being a substitute for human serum since this amount of HSA is the physiological value and it is the main component of human serum. The results for the five-point calibration

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Table 1 Calculation for the data analysis.

n

cfit,i

Mean value x¯ c of replica at concentration c (c = 1...m)

cfit =

Standard deviation  xc of replica n at concentration c (c = 1...m)

cfit =

Imprecision

imprecision =

Inaccuracy

inaccurcy =

Mean value of zero concentration response y¯ 0

S¯ fit (0) =

Standard deviation of zero concentration response y0

Sfit =

Limit of detection LOD

LOD = C ·

Lower limit of quantification LLOQ

LLOQ = C ·

with three-fold replicates lead to the standard curves depicted in Fig. 7 standard curves in 4% HSA. It is clearly demonstrated that the sandwich and binding inhibition assays work in parallel. Second item to be highlighted is the huge difference in LOD between the two assay formats. Compared to the sandwich assay for CRP performed in [11] the LOD of the binding inhibition assay for CRP could be shifted by at least a factor of 250 to higher concentration levels; from 0.35 ng/ml to 900 ng/ml. The clinically relevant range for CRP is between 1000 and 500,000 ng/ml. Therefore the calculated LOD at 900 ng/ml fits much better to the clinical relevant measurement range for CRP. The upper measurement limit could not be calculated but the sigmoidal curve is saturating at about 100,000 ng/ml, indicating an applicable measurement range for proper diagnostic statements. One big challenge for the sepsis diagnosis is to adjust the parameters in the clinically relevant range without working with different dilution factors for the different analytes. The aim is to have one diluted sample and measure all relevant parameters in parallel. The obtained result shows, that this is possible by combining the two assay formats. As it is seen the LODs of IL-6 and PCT are low compared to the LOD of CRP. PCT has a clinical region of interest between 0.05 and 50 ng/ml and IL-6 between 0.04 and 1.5 ng/ml, therefore a very low LOD is desirable. The achieved LODs for IL-6 and PCT are 0.27 ng/ml and 0.34 ng/ml respectively. Therefore these

i=1



n

1 n−1

n 

(cfit,i − cfit )

i=1 cfit

 ccfitfit n truec



i=1

2

· 100%



− 1 · 100%

Sfit (0)i

n

1 n−1



n 

2 (Sfit (0)i − S¯ fit (0))

i=1 A−D (S¯ fit (0)+3·Sfit )−D



 B1

−1

A−D (S¯ fit (0)+10·Sfit )−D

 B1

−1

Table 2 Imprecision and inaccuracy of standard curves in 4% HSA. Concentration CRP/IL-6/PCT [ng/ml]

Sorg imprecision CRP/IL-6/PCT

Sorg inaccuracy CRP/IL-6/PCT

100/0.5/0.5 1000/5/5 10,000/50/50 100,000/500/500

–/36/15 40/38/6 9/26/28 –/91/–

–/7/7 1/7/4 1/4/23 –//–

characteristics do not meet the clinical requirements yet, but PCT can be determined from the mid to the elevated clinical levels and both parameters at high elevated levels. Another import characteristic is the inaccuracy and the imprecision. Like described in [4] the mean value of the replicates should be both within 15% in the dynamic part of the calibration curves being between upper and lower limit of quantification. As it is seen in Fig. 7 and Table 2 the values for inaccuracy and imprecision are exceeding these specifications in some cases. But since these values are calculated for each set of replicates, some replicates meet the requirements. For IL-6 the replicates of 0.5 ng/ml shows 14% imprecision and 7% inaccuracy and the replicates at 5 ng/ml show 6% and 3% respectively both meeting clinical requirements. Additionally, CRP meets the requirements at 10,000 ng/ml with an imprecision of 9% and an inaccuracy of 1%. With further improvement of e.g. the chip fabrication and by implementing reference methods these results clearly indicate that it is possible to meet clinical requirements with a multiparameter assay for sepsis diagnosis with the presented system (see Table 3). .

Table 3 Imprecision and inaccuracy of standard curves in human plasma. Concentration IL-6/PCT/NPT [ng/ml]

Fig. 7. Standard curves in 4% human serum albumin. Standard curves of the sepsis parameters CRP (squares), PCT (circles) and IL-6 (rectangles) in 4 % human serum albumin solution. The x-axis shows the concentration of the corresponding analytes, the y-axis shows the analytes signals. Imprecision and inaccuracy are shown for each replicate.

Panel a 0.5/0.5/0.5 5/5/5 50/50/50 500/500/500 Panel b 0.5/0.5/1 5/5/10 50/50/100 500/500/1000

Sorg imprecision IL-6/PCT/NPT

Sorg imprecision IL-6/PCT/NPT

7/–/– 24/42/– 45/49/44 –/65/–

0.5/–/– 13/18/– 15/19/18 –/42/–

35/–/– 44/68/– 14/33/40 34/17/–

2/–/– 9/46/– 0.5/8/3 4/1/–

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of reproducibility especially at the concentration of 100 ng/ml. In general the experiments show a good accordance of the curve progression. To summarize it is clearly seen that the assay principle works for the parameters in human plasma. The results of this chapter and Section 3.1 show, that there are variances between the batches of chips and in many cases elevated values for the imprecision. Up to now deviations between the measurements are mainly based on batch to batch variations during the chip production. The chips for the presented standard curves are produced at very small scale. This implies that deviations of e.g. the planar waveguide chips itself, the coating process and the spotting are included in the presented characteristics of LOD, imprecision and inaccuracy. 3.3. Referencing method

Fig. 8. Standard curves in human plasma, test (a) and (b). Standard curves of the sepsis parameters NPT (squares), PCT (circles) and IL-6 (rectangles) in human plasma. The x-axis shows the concentration of the corresponding analytes, the y-axis shows the analytes signals. Imprecision and inaccuracy are shown for each replicate.

3.2. Standard curves in human plasma For further characterization of the system performance pooled human plasma was tested for the use as sample. Tests in human plasma are of high interest since this matrix is commonly used in hospitals. The results of two identical tests for PCT, IL-6 and NPT are depicted in Fig. 8 standard curves in human plasma, test (a) and (b). IL- 6 meets the clinical requirements at the concentration of 0.5 ng/ml by undergoing the 15% borderline for imprecision and inaccuracy for the three-fold replicate for test (a) and (b). For test (a) the LOD of IL-6 at 0.09 ng/ml fits more to the clinically relevant range of 0.04– 1.5 ng/ml, though test (b) has an increased value with a LOD of 0.77 ng/ml. PCT shows a higher LOD compared to the IL-6 assay. For test (a) PCT had a LOD of 1 ng/ml. Test (b) showed an increased signal of the zero sample value and its deviation and therefore PCT had a LOD at 37 ng/ml. But in general the two curves have a very high accordance and test (b) showed improved precision and accuracy. NPT was processed the first time in parallel with IL-6 and PCT. The clinically relevant range for NPT is from 2.5 ng/ml to >18 ng/ml [21] being not resolved in this test. In both tests the curves are just decaying at about 10 ng/ml. Test (b) obtained a higher degree

As described in Sections 3.1 and 3.2 multi-analyte measurements in a human matrix model (4% HSA), serum and plasma are feasible with the presented system. But the tests also showed a high deficit concerning the reproducibility of replicate measurements. Actually this deficit was not surprising since the amount of errors which can occur during the whole process including the signal detection is high. Errors in this process are composed by variances of the blank waveguide chip, chip coating, spotting, fluidic processing, immunoassay and read out. An idea to improve the characteristics for the imprecision and inaccuracy was the implementation of a reference method including the named errors in one signal. For this purpose a direct immunoassay was chosen to be used as reference reaction. A spotted reference antibody includes errors from the chip coating and spotting. A reference detection antibody with the same label as the detection antibodies for the sepsis parameters is mixed with the sample (dilution mix).The detection antibody reacts specific to the spotting antibody. Therefore the fluidic processing, the immunoassay and the read out are resolved. Since variances of the waveguide chip get mainly expressed by variances in the evanescent field and consequently in the fluorescent excitation this error is resolved as well. This means the signal of the reference assay comprises manifold variations and thus this signal can be used to correct the signals obtained from the sepsis assays spots. The requirement for this assay is a low cross reactivity with the other sepsis assays and with the human matrices. This method can be used to correct signals on chip by means of an intra-chip correction improving spot-to-spot variances and an inter-chip correction improving chip-to-chip variances. 3.4. Chip-to-chip correction In this chapter the chip-to-chip improvement due to the reference method will be shown. The calculation for this test is described in Section 2.8. The sepsis parameters NPT will be investigated in a binding inhibition assay and IL-6 in a sandwich assay format to cover all necessary assays for sepsis diagnosis. Both analytes were spiked in healthy human plasma samples. The sample in the NPT assay was diluted by factor 4 (plasma: diluting mix) and in the IL-6 assay by factor 10 and factor 4 respectively (plasma: diluting mix). Like before, each concentration was measured in triplicate. As seen in Fig. 9, the referencing method is working for both assay formats and for sepsis relevant parameters in a human plasma matrix. By comparing the imprecision (Table 4) for each chip replicate, the NPT assay improved by 9.8-fold and the Il-6 assay improved over 2.6-fold due to the reference method. The inaccuracy was also drastically reduced; for the NPT assay by 12.3-fold and for the IL-6 assay by 4.5-fold. The corrected signals show values of imprecision and inaccuracy below 20% for both assays. Additionally the LOD of the NPT assay was reduced from 9 ng/ml to 1 ng/ml, due to a lower variance of the zero samples. The LOD for the corrected

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Table 4 Imprecision and inaccuracy for original and corrected standard curves in plasma.

Concentration NPT [ng/ml] 4 20 100 1000 10,000 Concentration IL-6 [ng/ml] 0.48 2.4 12 60 300 3000

Sorg imprecision

Sorg inaccuracy

Sdot-ref imprecision

Sdot-ref inaccuracy

– 89 75 63 50

– 149 137 11 21

23 13 5 7 6

44 10 11 17 1

70 34 46 – 64 –

25 10 39 – 23 –

44 8 16 20 33 –

112 1.6 7.7 2.1 3.9 –

IL-6 assay was elevated from 0.3 to 0.6 ng/ml (LLOQ 1.1 ng/ml and 1.8 ng/ml), this is caused by the low signals given by the zero samples from a sandwich assay. But measurements above the LOD of the corrected IL-6 assay show a drastic reduction of the chip-tochip variances. The comparison of this test with the tests described in Sections 3.1 and 3.2 shows the following: The LOD of the IL-6 assay without referencing showed an LOD of 0.27 ng/ml in human serum substitute; an LOD of 0.09 ng/ml and 0.77 ng/ml in human plasma. This shows that the assay for the sepsis parameter is not influenced by the reference method since

Fig. 9. Original and corrected standard curves in plasma, test (a) IL-6 and (b) NPT. The x-axis shows the concentration of the corresponding analytes, the y-axis shows the analytes signals. Imprecision and inaccuracy are shown for each replicate. In (a) the original data (squares) and the corrected signals (circles) are shown for IL-6. For NPT in (b) the original data (squares) and the corrected signals (circles) are depicted.

the LOD shows very similar values. The quality of the NPT assay with reference method has been improved very drastically compared to the test without reference method. A low LOD and a very good reproducibility show the high quality of the binding inhibition assay. 3.5. Tests with clinical specimen The measurement with a serum substitute model or spiked human plasma is a good tool to characterize the system under realistic conditions. Nevertheless it is just a simulation. The measurement of plasma samples obtained from healthy patients and patients with elevated clinical parameters is a challenge which has to be taken by all systems intending to enter the POC routine. For this purpose the sepsis parameter IL-6 was chosen to be compared with the performance of established (not POC) clinical laboratory systems like the Siemens Immulite 2500 and the Cobas E411. Of course this test did not affect the diagnosis of any patient; it is a system comparison for an early-stage technical and clinical validation. For an extensive proof of principle the system compatibility to a clinical environment must be known. For this the performance in different clinical laboratories was tested. For this purpose the sepsis parameter IL-6 was tested in the university hospital of Tübingen (hospital 1) by using plasma samples and Munich (hospital 2) by using serum samples with the POC-System. First the results obtained with plasma samples will be explained. The concentrations of Il-6 plasma given by the Immulite 2500 were in pg/ml: 0.6, 68, 216, 400, 969, 1639, 5180 and 100,000. The result depicted in Fig. 10 shows the fitted measurements done with the system. Squares with error bars indicate triplicate measurements whereas single squares indicate single measurements. The x-axis depicts the concentration values measured by the Immulite system. Due to the low dilution factor interference factors were likely, leading in some cases to a more elevated inaccuracy than observed in the standard curves in the chapters before; please see Table 5. The measurements covered concentrations levels from < 1 pg/ml to > 100,000 pg/ml. The calculated LOD of the system was 6 pg/ml and a LLOQ of 76 pg/ml was achieved; for the calculation of the LOD the signals at concentration 0.6 pg/ml where used as zero sample. The calculated imprecision for the present triplicate measurements was in total below 25%. An inaccuracy between 5 and 182% was observed, whereas concentrations >1000 pg/ml had been below 20%. In the following the results obtained in clinical environment in hospital 2 will be described. In this case human serum samples were predetermined with the Roche Cobas E411. The concentrations were in pg/ml: 5.37, 77.91, 164.7, 227, 313.5, 1055, 5166 and 35,626 (see Table 6). In Fig. 11 the fitted result of the measurements of hospital 2 is shown. Again the x-axis shows the measured concentration

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Fig. 10. Patient plasma samples results for IL-6. The x-axis shows the concentration of the corresponding of the plasma IL-6 concentration being determined by established clinical laboratory systems. The y-axis shows the analytes signals. Imprecision and inaccuracy are shown for each replicate. If the replicate number is less than three, only the inaccuracy is calculated.

with the established clinical system: Cobas E411. And again single squares indicate single measurements the imprecision was only calculated if a triplicate measurement was present otherwise the inaccuracy of the single measurement was calculated. The values for imprecision above 1000 pg/ml are much more accurate than the ones below and values below 15% could be achieved. In general the inaccuracy is elevated, however, a clear signal to concentration correlation is present and the clinically relevant range is resolved. In Fig. 12 the obtained data of the two clinical laboratories is shown; in black the data of hospital 1 and in red the data of hospital 2. For a better comparison the signals are normalized to the mean value of lowest concentration signal of each test. The fitting of the data of hospital 1 is depicted in the figure. It seems as if the batch of chips was better fabricated for the hospital 1 test, which could explain the increased imprecision. As already explained, both data show increased inaccuracy and imprecision for IL-6 levels < 100 pg/ml being probably generated by interference factors (e.g. bilirubin). These factors are present in high concentrations, since the dilution factor is low at 0.75. Other measurements Table 5 Imprecision and inaccuracy for standard curve with patient plasma samples. IL-6 concentration

Sdot-ref imprecision

Sdot-ref inaccuracy

0.6 68 216 400 969 1639 5180

– 22 13 – – – 17

– 106 84 34/45 7/17 12 9

Table 6 Imprecision and inaccuracy for standard curve with patient serum samples. IL-6 concentration 5.37 77.91 164.7 227 313.5 1055 5166 35,626

Sdot-ref imprecision – 24 – – 56 9 1 –

Sdot-ref inaccuracy – 93 49/67 1 3 74 13 2/1

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Fig. 11. Patient serum samples results for IL-6. The x-axis shows the concentration of the corresponding of serum IL-6 concentration being determined by established clinical laboratory systems. The y-axis shows the analytes signals. Imprecision and inaccuracy are shown for each replicate. If the replicate number is less than three, only the inaccuracy is calculated; please see Table 6.

Fig. 12. Comparison of clinical IL-6 measurements. The x-axis shows the concentration of plasma (squares, hospital 1) and serum (circles, hospital 2) IL-6 concentrations being determined by established clinical laboratory systems. The yaxis shows the corresponding analyte signal. For a comparison the fit of the plasma standard curve is depicted.

with diluted samples by factor 4 and 10 (please see Section 3.4) show much more accurate values for imprecision and inaccuracy; but in general the data of the two experiments in two different hospitals with a time delay of two months show good accordance and results obtained with untreated patient samples. IL-6 could be resolved in the clinically relevant range, though it is a very low abundant protein at very low concentration levels in the range of pg/ml. This shows that a highly sensitive and fast assay can be processed automated and within 25 min by the system. 4. Conclusions The biochip based point of care system is capable to process a binding inhibition and a sandwich assay parallel and automated within 25 min. The system processes and detects multiparameter fluorescence assays for the sepsis relevant parameters IL-6, CRP, PCT and NPT. This assures a high diagnostic benefit for the patient, since the information of each analayte can be combined. By the

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combination of the binding inhibition and the sandwich low level LODs for IL-6 and PCT and a high level LOD for CRP can be obtained in parallel [19] being an important basis to work in the clinical relevant range. The parameter NPT is running in a binding inhibition assay because the molecule is too small to detect it in a sandwich assay format. Fluidic operations like diluting, mixing, metering, pre-incubating, incubating and washing were successfully implemented to reproduce a fluorescence immunoassay. The used sample volume is only 10, 25 or 75 ␮L. Together these points are meeting some important POCT criteria. The capability of the presented system was tested by spiking antigen standards into a buffer containing 4% human serum albumin (HSA). Standard curves of the three sepsis relevant parameters CRP, IL-6 and PCT could be obtained. IL-6 and PCT were additionally tested together with NPT in pooled human plasma showing the assay performance in a more complex human matrix. The curves show specific ranges and characteristics of the analytes detected by the system. The dynamic range of the assays covers for CRP the relevant range, for PCT mid and high elevated marker levels and for IL-6 high elevated levels in 4% HSA. By the use of two different assay formats a factor between 900 and 10 000 is between the LODs of the different analytes. For PCT and IL-6 in human plasma only elevated marker levels could be detected. At this point it has to be mentioned that the measured concentrations by the systems of the depicted curves and tables are actually ten-fold lower because of the dilution factor of ten. This dilution factor is beneficial to reduce cross-reactions however the LOD increases by this operation. Without the dilution factor PCT, IL-6 and NPT could match more convenient the clinical range. Several replicate values for accuracy and precision fulfil the clinical directive but in general these characteristics have to be reduced. For this purpose a referencing method is presented reducing imprecision and inaccuracy drastically. The comparison of referenced and non-referenced averaged data shows a reduction of impression of 2.6-fold for the IL-6 assay and 9.5-fold for the NPT assay respectively. The inaccuracy was could be reduced 12.3-fold for NPT assay and for the IL-6 assay by 4.5-fold. Thereby it can be stated that the reference method works for both binding inhibition and sandwich assay formats. The referenced signals show values of imprecision and inaccuracy below 20% for both assays fulfilling some important POCT criteria. Finally the platform was tested in two different hospital laboratories with patient samples of healthy and pathogen patients. The IL-6 levels of plasma and serum samples were determined by established laboratory devices. Due to a low dilution factor of 0.75 the low abundant protein IL-6 could be measured in clinical relevant ranges. Replicate measurements of concentrations >1000 pg/ml had imprecision’s below 20% and with the exception of one triplicate measurements an inaccuracy below 20% was achieved. Signals of concentrations < 1000 pg/ml had elevated values for the inaccuracy. These effects might be generated by interference factors and must be closer investigated. It could be shown, that the presented system measured fast low abundant sepsis relevant parameter in patient samples being a proof of principle for the clinical usability of the system. The presented investigations identified the difficulties for the intended use of the system and show the next steps to be taken.

Acknowledgements This work was funded by the ‘Health-CARE by biosensor Measurement And Networking’ (CARE-MAN) (NMP4-CT-2006-017333) research project supported by the European Commission. The authors thank the ethical committee of University of Tuebingen, Germany and Klinikum rechts der Isar, Germany, Drs. Petra

M. Krämer and Elisabeth Kremmer, both Helmholtz Centre Munich – German Research Centre for Environmental Health (GmbH), Germany and Milan Franek Veterinary Research Institute, Czech Republic for providing antibodies, Dr. Albrecht Pfäfflin, University of Tuebingen, Department of Internal Medicine, Clinical Chemistry unit, Germany and Prof. Dr. Peter Luppa, Klinikum rechts der Isar, Germany for supporting the patient sample measurements and Dr. Frantisek Skrob, Exbio, Czech Republic, for labelling the antibodies and several supporting information. We thank Bayer Technology Services for valuable support, particularly for providing us with planar waveguide chips. We thank Prof. Dr. Roland Zengerle, Department of MEMs Applications, Department of Microsystems Engineering (IMTEK), for support and helpful suggestions concerning the microfluidics. We thank Dr. Peter Koltay (Biofluidix) for providing the TopSpot spotter.

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Biographies Manuel Kemmler received his diploma degree in medical engineering 2006 at the University of Applied Sciences Furtwangen. He received PhD degree in 2011 at

M. Kemmler et al. / Sensors and Actuators B 192 (2014) 205–215 the University of Freiburg. He is working in the field of biotechnology including microfluidics and biosensing devices for medical diagnosis. Ursula Sauer is working in biochip related projects at ARC since 2001. She is experienced in all processes involved in development, production, optimization and validation of biochips. Her special interest is data analysis and quality control of both, data and methods. Erwin Schleicher received his Diploma degree in Chemistry in 1972 and his PhD degree in1974 both from the Technical University Munich. After his post-doctoral fellowship at the Dept. of Medicinal Chemistry, Purdue University, Indiana, USA, he joined the Institute of Clinical Chemistry and Diabetes Research Center at the Academic Hospital München-Schwabing and became the Director of this institution in 1991. In 1996 he moved to the University Hospital of Tübingen as head of the Clinical Chemistry department and Professor of Clinical Chemistry. His major scientific activities are the pathobiochemistry of the development of type 2 diabetes and its subsequent vascular complications, the molecular mechanisms of insulin signaling and miniaturization of diagnostic assays.

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Claudia Preininger studied chemistry at the Karl-Franzens University in Graz where she received her diploma (1993) and PhD degree in chemistry with distinction (1996). The focus of her thesis was the development of an optical sensor for biological oxygen demand and optochemical biosensors for detection of heavy metals. After research visits in Lund and Lisbon and post-docs in Pisa and Florence she has been with the Austrian Research Centers GmbH – ARC since 1999. She is currently project manager in the Department of Bioresources and responsible for protein chip surface and assay development. Albrecht Brandenburg received the diploma degree in physics 1983 from University of Münster, Germany and the PhD degree in 1989 in Engeneering from University of Clausthal. Since 1984 he is with Fraunhofer Institute of Physical Measurement Techniques in Freiburg, Germany. He worked in the field of integrated optical technology for applications in telecommunication and physical sensing. For ca. 10 years he is developing optical systems for biosensing and medical diagnosis including e.g. biochip readout technologies, label free detection and automated cell monitoring. Since 2002 he is also member of University of Freiburg, teaching in the field of microsystem technology.