Method for Label-Free Detection of Femtogram Quantities of Biologics in Flowing Liquid Samples

Abstract

Rapid (∼10 min) measurement of very low concentration of pathogens (∼10 cells/mL) and protein (∼fg/mL) has widespread use in medical diagnostics, monitoring biothreat agents, and in a broader context as a research method. For low-level pathogen, we currently use culture enrichment methods and, thus, rapid analysis is not possible. For low protein concentration, no direct method is currently available. We report here a novel macrocantilever design whose high-order resonant mode near 1 MHz exhibits mass detection sensitivity of 10 cells/mL for cells and 100 fg/mL for protein. The sensor is 1 × 3 mm and uses a piezoelectric layer for both actuation and sensing resonance. Sample is flowed (∼1 mL/min) past the antibody-immobilized sensor, and as antigen binds to the sensor, resonance frequency decreases in proportion to antigen concentration. The sensor showed selectivity to the pathogen even though copious nonpathogenic variant was simultaneously present.Rapid and single-step label-free direct detection of proteins in flowing liquid samples at a concentration of 100 fg/mL has not been reported to date. Neither has there been any report on detecting pathogens at 10 cells per mL from samples of tens of milliliters under flow conditions. Both measurements have significant applications in medicine (for biomarkers in body fluids), 1 environmental monitoring (pathogens in drinking water), food safety (Listeria, 2 Cyrptosporidium, Giardia, 3 and Escherichia coli poisoning 4 ), and biodefense (biothreat agents). In this paper, we show designs of millimeter-sized cantilever sensors that exhibit mass change sensitivity of femtograms under liquid flow conditions and that are potentially useful in the applications mentioned above. Three types of experiments are reported to demonstrate the high detection sensitivity.Cantilever biosensors have attracted considerable interest in the past decade for label-free detection of proteins and pathogens because of their promise of very high sensitivity. 5,6 Excellent reviews have appeared that summarize progress. 7,8 Briefly, the binding of an antigenic target to an antibody-immobilized cantilever surface changes the cantilever's surface stress resulting in a deflection response. 9,10 Cantilever biosensors 11,12 have been successfully used in DNA hybridization studies, 9,13 detection of known cancer proteins, 14 environmental and foodborne pathogens, 15,16 biomarkers, 17,18 and explosives. 19 In dynamic mode, the attachment of antigen causes a resonant frequency decrease because of increase in mass. 7 Magnitude of bending deflection can be monitored by various transduction mechanisms. 7,[20][21][22] Because significant damping occurs in the dynamic mode, static deflection method is preferred when continuous measurement under liquid immersion is needed. When measurement in liquid flow condition is required, the bending mode becomes noisy and less trustworthy because of fluctuating hydrodynamic forces. It is well established 23 that for the dynamic method to provide reasonable signals, c...

Nanomechanical resonators and their applications in biological/chemical detection: Nanomechanics principles

“…Until now, with few exceptions [22,23,172], there have been very few studies on in situ detection of proteins in aqueous environments. Here, in situ detection of proteins in solution is of high significance, because in situ detection allows the real-time monitoring of protein-protein interactions, which will enable novel insights into the kinetics of protein-protein interactions.…”

Online Portable Microcantilever Biosensors for Salmonella enterica Serotype Enteritidis Detection

“…Ilic et al (2001) first reported the detection of single Escherichia coli (EC) cells using a cantilever array vibrating in vacuum. Maraldo et al (2007) extended the work of Ilic and co-workers, developing a composite, self-excited lead zirconate titanate (PZT)-glass millimeters size cantilever coated with anti-EC antibodies to detect the pathogen, EC O157:H7. They recently obtained impressive sensitivities for measurements both in buffered salt solutions (10 cfu/mL) and ground beef (100 cfu/mL).…”

A Fast Room-Temperature Poling Process of Piezoelectric Pb(Zr<SUB>0</SUB><SUB>.45</SUB>Ti<SUB>0</SUB><SUB>.55</SUB>)O<SUB>3</SUB> Thin Films

“…We fitted P r (Fig. 4(a)) with an exponential function of the form P r t pol HT = P r0 + P r 1 − exp −t pol /t s400 (1) Here P r0 is the remnant polarization value before poling, P r the maximum achievable change in remnant polarization by the polarization process, and t s400 the time constant for poling with a dc poling field of 400 kV/cm. We obtain the fitting parameters as given in Table II for the SG(001) and the Text(001) films.…”

“…Sensing and actuation capabilities in most of the MEMS devices utilize the piezoelectric effect, such as in bio-sensors, [1][2][3] micro-machined ultrasonic transducers (MUTs), 4 5 accelerometers 6 resonators, 7 and micro-pumps. 8 9 Among piezoelectric thin-film materials, Pb(Zr x , Ti 1−x O 3 (PZT) films can offer an attractive option for piezoMEMS technology due to their superior ferroelectric and piezoelectric properties, and moreover, ease of integration into microsystems.…”