Surface-enhanced Raman spectroscopy (SERS) of living cells has rapidly become a powerful trend in biomedical diagnostics. It is a common belief that highly ordered, artificially engineered substrates are the best future decision in this field. This paper, however, describes an alternative successful solution, a new effortless chemical approach to the design of nanostructured silver and heterometallic continuous coatings with a stochastic ''coffee ring'' morphology. The coatings are formed from an ultrasonic mist of aqueous diamminesilver hydroxide, free of reducing agents and nonvolatile pollutants, under mild conditions, at about 200-270 C in air. They consist of 30-100 micrometer wide and 100-400 nm high silver rings composed, in turn, of a porous silver matrix with 10-50 nm silver grains decorating the sponge. This hierarchic structure originates from ultrasonic droplet evaporation, contact-line motion, silver(I) oxide decomposition and evolution of a growing ensemble of silver rings. The fabricated substrates are a remarkable example of a new scalable and low cost material suitable for SERS analyses of living cells. They evoke no hemolysis and reduce erythrocyte lateral mobility due to suitable ''coffee ring'' sizes and a tight contact with the silver nanostructure. A high SERS enhancement, characteristic of pure silver rings, made it possible to record Raman scattering spectra from submembrane hemoglobin in its natural cellular environment inside single living erythrocytes, thus making the substrates promising for various biosensor chips.

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Hypoxia of various origin and localization is accompanied by changes in some physical and chemical properties of erythrocytes: deformability, plasma membrane viscosity, and the oxygen-binding capacity of hemoglobin [1,[4][5][6]. Under the conditions of brain ischemia, these properties are studied insufficiently. After postischemic reperfusion (PIR) that restores blood circulation, the oxygen partial pressure in plasma increases, which may stimulate the generation of reactive oxygen species (ROS) and affect the erythrocyte functions. In blood plasma, Cu,Zn-superoxide dismutase (SOD) and ceruloplasmin (CP) are involved in utilization of superoxide anion radical ( ) that triggers ROS formation. In this study, changes in the viscosity of erythrocyte plasma membrane and the é 2 -binding ability of hemoporphyrin of deoxyhemoglobin were studied. SOD activity and CP level were also measured in blood plasma of rats with brain ischemia before and after brain PIR.White outbred male rats weighing 272 ± 11 g were used in experiments. The animals were divided into three groups: the sham-operated rats (the control) ( n = 10), the rats with brain ischemia ( n = 10), and the rats with postischemic brain reperfusion ( n = 7). One day before the experiment, both carotids of the anesthetized animals were underpinned with a fishing line (0.3 mm) that was later withdrawn under skin through the polyethylene tubes into the interscapular region. After one day, a one-stage complete occlusion of both carotids was induced by carotid retraction into the tubes by means of the fishing line; subsequent release of carotids led to PIR. Blood samples (3 ml of blood mixed with heparin, 10 U/ml) were taken from the jugular veins of O 2 -the sham-operated rats, the second group rats 2 h after ischemia, and the third group rats 2 h after ischemia that was followed by 30-min PIR.The viscosity of erythrocyte plasma membrane was determined by the method of electron paramagnetic resonance (EPR) on an RE-1308 EPR spectrometer (Russia). The spin-labeled stearic acid analogues, 5-and 16-doxylstearic acids (5-DS and 16-DS), which have a paramagnetic fragment in positions differing with respect to the carboxylic group, served as a spin probe. With these reagents, the orderliness of fatty-acid phospholipid tails can be tested at different depth from the membrane surface (0.6-0.8 nm and about 2.2 nm). The following parameters were calculated from the EPR spectra: (1) the orderliness parameter S for the 5-DS probe, which characterizes the acyl-chain mobility and (2) the correlation time of the nitrooxyl radical rotation τ for the probe 16-DS [3].Changes in the conformation of hemoglobin hemoporphyrin were estimated by combination scattering (CS) spectroscopy [8]. The blood sample in HEPES-Hank's solution (ratio, 1 : 4, pH 7.4-7.5) was placed into a hermetically closable capillary that was fastened in the holder of the device. We used a DFS-24 monochromator (Russia) with excitation from the solid-state Nd-laser ( λ = 473 nm, R = 18-20 mV) and a FEU-79 photon-recor...

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