Ewelina
Wajs‡
*a,
Girish
Rughoobur
b,
Keith
Burling
c,
Anne
George
d,
Andrew J.
Flewitt§
a and
Vincent J.
Gnanapragasam§
cdef
aElectrical Engineering, University of Cambridge, 9 JJ Thomson Avenue, Cambridge, CB3 0FA, UK. E-mail: ewelina.wajs@icfo.eu
bMicrosystems Technology Laboratories, Massachusetts Institute of Technology, 60 Vassar Street, Cambridge, MA 02139, USA
cNIHR Core Biochemical Assay Laboratory (CBAL), Cambridge University Hospitals NHS Foundation Trust, UK
dCambridge Urology Translational Research and Clinical Trials, Addenbrookes Hospital, UK
eAcademic Urology Group, Department of Surgery, Cambridge University Hospitals NHS Foundation Trust and University of Cambridge, UK
fCRUK Cambridge Cancer Centre, Cambridge Biomedical Campus, University of Cambridge, UK
First published on 15th April 2020
Easy monitoring of prostate specific antigen (PSA) directly from blood samples would present a significant improvement as compared to conventional diagnostic methods. In this work, a split mode thin film bulk acoustic resonator (TFBAR) device was employed for the first time for label-free measurements of PSA concentrations in the whole blood and without sample pre-treatment. The surface of the sensor was covalently modified with anti-PSA antibodies and demonstrated a very high sensitivity of 101 kHz mL ng−1 and low limit of detection (LOD) of 0.34 ng mL−1 in model spiked solutions. It has previously been widely believed that significant pre-processing of blood samples would be required for TFBAR biosensors. Importantly, this work demonstrates that this is not the case, and TFBAR technology provides a cost-effective means for point-of-care (POC) diagnostics and monitoring of PSA in hospitals and in doctors’ offices. Additionally, the accuracy of the developed biosensor, with respect to a commercial auto analyser (Beckman Coulter Access), was evaluated to analyse clinical samples, giving well-matched results between the two methods, thus showing a practical application in quantitative monitoring of PSA levels in the whole blood with very good signal recovery.
Prostate cancer is one of the few cancers with a known biomarker used for screening, diagnosis and monitoring after treatment.4 PSA is a 34 kDa glycoprotein expressed predominantly by the prostate gland and present in large amounts in its tissue and semen. Although, PSA is not prostate specific and it can be present in other body fluids and tissues in healthy men as well as in women, it is still a very important biomarker in prostate cancer diagnostics.5 In female ejaculate PSA levels are almost as high as in male, but its high concentrations can further be found in breast milk and amniotic fluid. PSA is also present in the serum of women with breast, lung, or uterine cancer and in some patients with renal cancer. Thus, PSA as a single biomarker cannot be always directly linked to prostate cancer only, but undoubtedly testing for its elevated levels in the blood (>4 ng mL−1) can increase the chances for finding cancer in its earliest, most curable stage.6 Most importantly, by monitoring levels of PSA in the blood indicates how well cancer treatment has worked and it helps significantly in further cancer treatment and prognosis. Typically, PSA should drop to very low levels after surgery or radiation treatment for prostate cancer.7 Despite PSA testing being already widely available, it still largely relies on sample processing in clinical laboratories, which makes it impractical and too expensive for many applications. Thus, the need for a POC self-testing system has led to constant development of novel detection strategies that are suitable for miniaturisation as well as easy, rapid and accurate PSA measurements. Many different types of detection systems for PSA analysis from serum or blood samples have been previously reported in the literature, i.e. optical, electrochemical or mass-based methods.8–13 However, these are yet to be implemented in a regular POC test for rapid and inexpensive monitoring of PSA levels that is clinically accepted. They also tend to give PSA ranges rather than absolute values and are more used for detection of high levels rather than give granularity on actual measure levels. This is crucial if the disease is to be monitored accurately. Some commercially available PSA testing kits include home screening tests e.g. the SELFCheck® or PRIMA®, and lab testing kits, such as ELISA. However, most hospital laboratories measure total PSA by 2-site immunoassay on large automated analysers (Siemens Centaur, Beckman Coulter Access, Roche Elecsys, etc.). An important issue is how well these tests work in the context of a POC or patient administered test i.e. measurements made using whole human blood from a pin prick sample.
TFBAR gravimetric sensors can recognise biological species by detecting a very small change in mass attached to their sensing surface. A general structure of this device is presented in the Fig. 1; the device fabrication and operation was described in detail elsewhere.14 The essence of the device is that two resonant frequencies are produced in a single structure. The first resonator (R1) with an anti-PSA antibody modified gold surface responds to the biomolecule (antigen) attachment, whilst the second resonator (R2) without the gold layer is not sensitive to it. Yet, they both respond to any other influences (e.g. temperature, humidity). This means that the difference between these two resonant frequencies Δ(f2 − f1) can be used to quantitatively measure the target analyte specifically from a sample. Key features of TFBAR devices include their very small size (the sensing area is typically ∼100 × 100 μm), low power consumption, ease of multiplexing on a single, small chip and low cost.15 These characteristics make TFBAR devices very attractive for POC immunotesting.16
In general, there are many difficulties that various sensing systems still need to overcome, when using a fresh whole blood samples for testing, even the existing commercial finger prick tests for blood glucose monitoring in diabetics. Whole blood has one of the most complex matrices as compared to other body fluids, containing many various matrix components that can easily affect response of bioanalytical processes. Most notably, measurements performed on real samples from patients often create major difficulties in sample handling as well as data interpretation due to patient/to/patient variability and a multitude of interfering analytes that need to be either removed or ignored by a selective enough sensor. There are often major sample preparation steps involved in removing interfering entities from the sample, such as red and white blood cells or lipids.17–20 Therefore, it has widely been believed that non-specific surface interactions would mean that also TFBAR devices would require complex fluids, like blood, to be pre-processed for successful biosensing. This would be rendered impractical for POC testing and it would result in increased associated costs. In this work, a previously reported acoustic split mode resonator has been used for the first time for direct measurements of PSA levels in whole blood from patients in a hospital setting. To demonstrate a proof-of-concept for this novel detection method based on TFBAR technology, a simple bioassay for total PSA detection was used. This type of biosensor demonstrated high sensitivity and low LOD and most importantly, it was done without sample pre-processing. Results were compared with those from the commercial Beckman Coulter Access analyser.
For the TFBAR detection system, a small volume (10 μL) of either PSA in buffered solution or a fresh blood sample (from 7 patients) was spotted on the antibody-modified gold active surface and left to incubate for 5 min and 15 min in the humidity chamber, respectively (section 2.2, ESI†). These allowed the recognition of the target protein by the immobilised receptors. All samples were taken as part of an ethically approved study (Ethics 03/018, DIAMOND national study) in our centre with informed consent obtained from all participants. All experiments were performed in compliance with relevant laws or guidelines of Cambridge University and approved by the NRES Committee East of England, UK. After extensive rinsing with MilliQ water and drying with nitrogen, the devices were measured using 150 μm pitch ground-signal-ground (GSG) probes connected to a network analyser (NA-E5062A, Keysight Technologies, Santa Rosa, CA, USA), see sections 1 and 2, ESI.† To assess the reproducibility, the measurements were repeated 5 times for each sample concentration. The devices were cleaned with an argon plasma using a rf (13.56 MHz) Reactive Ion Etching (RIE) tool and checked for its reusability (Fig. S3, ESI†).
The great advantages of the TFBAR technology over other conventional methods are: (i) it is simpler and less costly compared to ELISA-type assays (i.e. Access immunoassay), (ii) it does not require additional labelling for the detection, (iii) it can be performed in a single reaction without additional reagents and (iv) it is compact and therefore, portable.
The TFBAR measurements (Δ(f2 − f1)) were performed using a portable network analyser (FieldFox, Model: N9913A from Keysight Technologies) and the unknown concentrations of PSA antigen in the whole blood samples were determined using the calibration plot obtained from the buffered solutions (Fig. 2B). The results acquired from both methods (TFBAR detection and commercial Access immunoassay) are summarised in the Table 1.
Sample from patients | TFBAR biosensor (ng mL−1 ± SD) | TFBAR biosensor (% RSD) | Access immunoassay (ng mL−1) |
---|---|---|---|
1 | 1.81 ± 0.86 | 47.5 | 0.47 |
2 | 4.20 ± 0.35 | 8.3 | 3.25 |
3 | 4.50 ± 0.70 | 15.5 | 3.90 |
4 | 6.00 ± 0.86 | 14.3 | 5.29 |
5 | 6.30 ± 0.51 | 8.0 | 5.70 |
6 | 6.40 ± 0.82 | 12.8 | 5.73 |
7 | 9.15 ± 0.42 | 4.5 | 9.60 |
The obtained values of PSA concentration in blood were in majority higher from TFBAR sensor than those obtained by Access immunoassay. Also, the reproducibility of the TFBAR responses compared to those from buffered solutions showed higher RSD values (up to 15.5%) for five independent experiments (from 1 to 10 ng mL−1). At concentrations below 1 ng mL−1 the TFBAR sensor showed very high RSD of 47.5%, however this does not have high clinical impact as in general, PSA levels that are below 4.0 ng mL−1 are considered as normal. Furthermore, these differences in the measurements may be due to insufficient blocking of the surface of the TFBAR sensor to prevent all non-specific interactions from a complex matrix such as whole blood and/or insufficient washing cycles of the device after the last step of incubation with the whole blood samples. Additionally, the use of polyclonal antibodies for capture could increase false positives due to sample contamination.
It may be possible to achieve even higher performance of the TFBAR sensor through further optimisation of its surface and immobilisation techniques. Our recent findings indicate that the sensitivity of the split mode TFBAR sensor depends on the thickness and roughness of the vacuum-deposited gold film. However, it also depends on the uniformity of the immobilisation layer and cleanliness of the sensing surface prior to functionalisation.21,22 It should be noted that whilst high sensitivity of 101 kHz mL ng−1 was achieved for PSA measurements in buffered solutions, the sensitivity in actual human whole blood samples decreased to 80 kHz mL ng−1. This could be due to the fact that analyte detection in a complex matrix such as blood is extremely difficult. Since blood contains thousands of various competing biomolecules and many of them at much higher concentrations than the target analyte, there is a high probability of false positives due to binding of the capture antibody to a non-target molecule that has a similar structural motif.
The possible effect of the serum matrix on target detection using the TFBAR biosensor was also studied by spiking blood samples with different amounts of PSA antigen (Table S1, ESI†). As the signal recovery was close to 100% it indicated the applicability of the system to the analysis of a real clinical samples.
Consequently, more work is needed to develop an integrated microfluidic system, which can further improve the performance of the TFBAR biosensor. Making this system fully automated with embedded electronics can minimise or even eliminate the “human error” during the experiment. The development of additional receptors and immobilisation methods for multiplexed assays could also help to improve the detection reproducibility of the TFBAR device. However, it is notable that the difference in TFBAR readings and Access immunoassay is in the sub 1 ng mL−1 range which is a very acceptable error margin in the context of utility in clinical practice. Particularly so, as PSA readings are known to fluctuate with normal biological variations.24 Alternatively, a standard correction can be applied to TFBAR reading, and so a future POC test could have an error of 1 ng mL−1 built in its system.
Footnotes |
† Electronic supplementary information (ESI) available: TFBAR device characteristics, materials used in this work and assay optimisation. See DOI: 10.1039/d0nr00416b |
‡ Present address: ICFO, Mediterranean Technology Park, Av. Carl Friedrich Gauss 3, 08860 Castelldefels (Barcelona), Spain. |
§ Joint senior authors. |
This journal is © The Royal Society of Chemistry 2020 |