Biomimetic receptors for bioanalyte detection by quartz crystal microbalances - from molecules to cells

Abstract

A universal label-free detection of bioanalytes can be performed with biomimetic quartz crystal microbalance (QCM) coatings prepared by imprinting strategies. Bulk imprinting was used to detect the endocrine disrupting chemicals (EDCs) known as estradiols. The estrogen 17β-estradiol is one of the most potent EDCs, even at very low concentrations. A highly sensitive, selective and robust QCM sensor was fabricated for real time monitoring of 17β-estradiol in water samples by using molecular imprinted polyurethane. Optimization of porogen (pyrene) and cross-linker (phloroglucinol) levels leads to improved sensitivity, selectivity and response time of the estradiol sensor. Surface imprinting of polyurethane as sensor coating also allowed us to generate interaction sites for the selective recognition of bacteria, even in a very complex mixture of interfering compounds, while they were growing from their spores in nutrient solution. A double molecular imprinting approach was followed to transfer the geometrical features of natural bacteria onto the synthetic polymer to generate biomimetic bacteria. The use of biomimetic bacteria as template makes it possible to prepare multiple sensor coatings with similar sensitivity and selectivity. Thus, cell typing, e.g., differentiation of bacteria strains, bacteria growth profile and extent of their nutrition, can be monitored by biomimetic mass sensors. Obviously, this leads to controlled cell growth in bioreactors.

Figure 1.

Chemical structures of estradiol (E2), ethinylestradiol (EE2) and estradiol benzoate (E2B).

Figure 2.

Sensor effects against E2 as a function of phloroglucinol content in polyurethane.

Figure 3.

Sensor effects as a function of β-estradiol concentrations after incorporating porogens (pyrene, diphenylmethane) in molecular imprinted polyurethane during synthesis.

Figure 4.

Sensor responses of estradiol imprinted polyurethane against its templated analyte (estradiol). Non-imprinted polyurethane layer served as reference for differential measurements.

Figure 5.

Selectivity pattern between β-estradiol (E2), and its structural analogues such as estradiolbenzoate (E2B) and ethinylestradiol (EE2). Sensor responses were also obtained by exposing to environmental estrogen, bisphenol A, which also affects the hormone system.

Figure 6.

Polyurethane sensitive layer having cavities for reversible inclusion of E. coli, imprinted with a synthetic bacteria stamp (replica of E. coli W) made of silicon (a) 2-dimensional presentation of the surface with AFM—contact mode; (b) Section analysis: depth profile of the indicated cavities in figure (a); (c) 3-dimensional presentation of the polymer section from figure (a), graphical processing of the results with the program WSxM.

Figure 7.

Concentration dependent frequency response of polyurethane layer imprinted with E. coli strain W replica (1 mg of bacteria approximately corresponds to 5 × 10⁸ cells).

Figure 8.

Cross sensitivity of a E. coli bacteria sensor, 10 MHz QCM coated with polyurethane and imprinted with synthetic E. coli W and B stamps sensitive to strain W, and strain B, respectively. E. coli B and W having a concentration of 5 mg/mL in water were measured while exposing to different sensors at room temperature.

Figure 9.

Surface characterization of Bacillus subtilis spores by atomic force microscopy in contact mode.

Figure 10.

The transformation of Bacillus subtilis spores into the respective bacteria analyzed by light microscopy.

Figure 11.

Measuring the growth of Bacillus subtilis from its spores by B. subtilis imprinted polyurethane, in nutrition solution of 2% ammonium sulfate and 10% glucose at temperature 42 °C and pH 7.2.