is a major pathogen for humans. It is a common cause of infections, from minor ones such as abscesses and sinusitis to life-threatening diseases such as bacteremia, endocarditis and sepsis [1
]. Its antibiotic-resistant strains, e.g., methicillin-resistant S. aureus
(MRSA) are a serious problem in healthcare [2
]. Besides being the origin of hospital-acquired infections, S. aureus
produces seven different toxins that cause food poisoning [3
]. It is generally accepted, that 105
cells per g of food produce sufficient enterotoxins to cause food poisoning [6
Since the relevance of this pathogen was discovered, many approaches in the development of rapid detection methods for infection control were investigated, as reviewed by Law et al. [7
] and Zhao et al. [8
]. According to these reviews, traditional methods, such as plate counts using selective agar, convince with their simplicity, low costs and high accuracy but take 4 to 6 days to yield results. Nevertheless, they are still regarded as the gold standard. One promising alternative method is polymerase chain reaction (PCR). The commercially available Xpert MRSA assay (Cepheid International, Sunnyvale, CA, USA) for example requires 2 h from DNA extraction to assay result [9
]. However, complex sample preparation by trained staff is needed.
According to Zhao et al., the most rapid detection methods are based on biosensor technology. Biosensors are devices, which use biological components as recognition elements to provide specific affinity to the desired target. The recognition element is coupled to a transducer, which transforms the biological into an electrical signal [10
]. To be commercially successful, a biosensor has to meet several requirements, e.g., low cost, fast response and high sensitivity. Therefore, despite its complexity, many researchers recognize the high potential of electrochemical impedance spectroscopy (EIS).
EIS is a fast label-free technique to measure the properties of electrode surfaces and bulk electrolytes. Owed to the progress in engineering and electronics during the last decades, high performance miniaturized impedance instruments are available for a relatively low budget [11
]. EIS was used successfully for biosensors with various recognition elements [12
]. For example, Bekir et al., developed an electrochemical immunosensor using antibodies against S. aureus
]. They report a detection limit of 10 CFU·mL−1
of S. aureus
, exploiting the impedance change of the electrode surface caused by the affinity reaction of the immobilized antibodies.
To overcome the limitations of antibodies, such as high manufacturing costs, instability to high temperatures and short shelf life, aptasensors employ aptamers as recognition element [15
]. Aptamers are synthetic, single-stranded nucleic acid molecules that can fold into complex three-dimensional structures allowing them to bind targets based on structure recognition with high affinity and specificity. They are selected using the SELEX procedure (systematic evolution of ligands by exponential enrichment), an iterative in vitro selection and amplification method [16
Electrochemical aptasensors were reviewed by Willner et al. [17
]: besides the well-known thrombin aptamer [18
], other impedimetric aptasensors emerged ranging from the detection of potassium ions [19
] and small molecules, such as ethanolamine [20
], to whole cells, e.g., Salmonella typhimurium
]. Shahdordizadeh et al., provided a review of recent advances in optical and electrochemical aptasensors for the detection of S. aureus
]. They report on aptamers selected against staphylococcal toxins, staphylococcal teichoic acid, staphylococcal protein A and S. aureus
as whole bacteria. The indirect detection of S. aureus
via aptamers targeting the toxins excreted by the pathogen are limited due to the difficulty in correlation of the sensor signal to the presence of viable microorganisms. Therefore, direct detection is favored. In the field of optical aptasensors, fluorescence is most prominent, but also one colorimetric aptasensor was developed [23
]. Using dielectrophoretic enrichment and fluorescent nanoparticles, Shangguan and coworkers developed an optical aptasensor with a limit of detection (LoD) of 93 CFU·mL−1
and an assay time of 2 h [24
]. By the use of upconversion nanoparticles, the fluorescence intensity was increased and Duan et al., gained a LoD of 8 CFU·mL−1
]. Chang et al., developed an optical aptasensor for the single cell detection of S. aureus
within 1.5 h [26
]. The detection principle is based on resonance light scattering of modified gold nanoparticles. Optical sensors have the disadvantage that complex biological samples often interfere with the detection process. Furthermore, electrochemical methods are appreciated for their fast response time, higher sensitivity, low-cost fabrication, simple automation and lower sample volumes. In their review, Shahdordizadeh et al., described five electrochemical aptasensors for the detection of S. aureus
]: Two are based on potentiometry with LoDs of 800 CFU·mL−1
] and single cell detection [28
]. Another used voltammetry to reach a LoD of 1 CFU·mL−1
] and Lian et al., combined interdigital electrodes (IDE) with quartz crystal sensor to detect the bacteria as low as 12 CFU·mL−1
]. Jia et al., used a glassy carbon electrode with aptamer modified gold nanoparticles to impedimetric detect a lower limit of 10 CFU·mL−1
within 60 min [31
All mentioned optical and electrochemical aptasensors used different aptamers, but have in common, that the aptamers were selected in a Cell-SELEX, wherein whole cells were used as target for aptamer generation. Although purposive, this has the disadvantage that it stays unknown, which part of the cell surface is targeted by the aptamer. Thus, it is also unknown, which S. aureus
strains can be bound by these aptamers. S. aureus
is known for its ability to adapt its genetics quickly to new environments. Nevertheless, the conserved sequence of the immune-evasive factor protein A shows only one mutation in 70 months [32
]. The surface bound protein A enhances S. aureus
’ adhesion to wounds by binding to the von Willebrand factor (vWF) and prevents phagocytosis by binding to the Fc region of various immunoglobulins [33
]. Protein A is bound to peptidoglycans on the cell wall of S. aureus
and not found on other bacteria. Therefore, protein A is an excellent target for the detection of S. aureus
cells. Also in PCR methods, the spA
gene, encoding protein A, is used to distinguish between S. aureus
and other bacteria.
A DNA aptamer targeting staphylococcal protein A was selected by the FluMag-SELEX procedure in 2015 [34
]. This aptamer development aimed to detect intact bacterial cells of S. aureus
via the protein A bound to its cell surface. Binding characteristics of the aptamer to protein A were studied intensively by different methods such as bead-based fluorescent binding assay, surface plasmon resonance (SPR), microscale thermophoresis (MST), and enzyme-linked oligonucleotide assay (ELONA) [35
The structural features of an aptamer play a major role in biosensor development. In case of the protein A-binding aptamer, a combination of two structural elements is important for its functionality: First, an intact and free 5′-end, folding into an imperfect stem-loop motif, is crucial for binding to protein A. Second, the aptamer folds into a parallel G-quadruplex structure as demonstrated by circular dichroism spectroscopy [36
In the present study, we developed a biosensor detecting Staphylococcus aureus
by its surface bound protein A, which is highly conserved and only found on S. aureus
. The protein A-binding aptamer served as biological recognition element. In combination with electrochemical impedance spectroscopy as measurement method, rapid and label-free detection was achieved. By immobilization of thiol-modified aptamer on gold electrodes by self-assembly, binding of S. aureus
was detected in a flow-through chamber with a three-electrode setup in buffer solution containing ferri-/ferrocyanide. Upon binding of S. aureus
, the impedance increased due to the hindrance of the electron transfer between ferri-/ferrocyanide and the electrode surface (Figure 1
). Herein, we elucidate the development of an impedimetric aptasensor and present novel insights on the use of aptamer-based electrochemical biosensors for the rapid and selective detection of S. aureus
This study provides the proof of principle for an impedimetric biosensor for the rapid detection of S. aureus, based on the protein A-binding aptamer. Successful co-immobilization of protein A-binding aptamers and 6-mercapto-1-hexanol on gold electrodes resulted in an average density of 1.01 ± 0.44 × 1013 aptamers per cm2. The immobilization density can be influenced by the ratio of aptamer to 6-mercapto-1-hexanol (MCH) as shown with chronocoulometry. We showed with MST measurements that ferri-/ferrocyanide, necessary as redox couple for faradaic impedance measurements, has no significant influence on the binding of the aptamer to its target. The biosensor displayed sensitive binding to protein A with a KD of 18.5 ± 1.8 nM and a LoD of 3 nM. Our results also showed the excellent selectivity of the developed sensor, with signals below LoD upon exposure to high concentrations of the functionally similar proteins G and L.
When exposed to live S. aureus cells, our developed aptamer-based biosensor showed a KD of 111 ± 96 CFU·mL−1 and a LoD of 10 CFU·mL−1, which is in good agreement with other reported assays or sensors. Our results also prove the high selectivity of the aptamer, distinguishing between S. aureus and protein A- deficient bacteria, such as E. coli and S. epidermidis.
For application in a clinical setting, an additional step for the evaluation of the different detectable S. aureus strains and their possible antibiotic resistance (e.g., by PCR) may have to be considered. Furthermore, the influence of different ionic strength buffers and sample matrices on the biosensor performance have to be investigated closely.
This work demonstrated that the protein A-binding aptamer can be used as recognition element in impedimetric aptasensors for successful, rapid, sensitive and selective detection of S. aureus in buffer. It contributes to the deeper understanding of impedimetric aptasensors and their development. We provided a fundamental base for inexpensive and robust biosensing, utilizing aptamer receptors. The advantages of using gold electrodes are their robustness, enabling regeneration and subsequent reuse of the biosensor. The simplicity of our design enables easy reproduction and the developed microfluidic system can be easily automated. Furthermore, combination with electrode patterning may enable the parallel measurement of multiple analytes when functionalized with different aptamers, in the future.