Characterization of cellular chemical dynamics using combined microfluidic and Raman techniques

The integration of a range of technologies including microfluidics, surface-enhanced Raman scattering and confocal microspectroscopy has been successfully used to characterize in situ single living CHO (Chinese hamster ovary) cells with a high degree of spatial (in three dimensions) and temporal (1 s per spectrum) resolution. Following the introduction of a continuous flow of ionomycin, the real time spectral response from the cell was monitored during the agonist-evoked Ca2+ flux process. The methodology described has the potential to be used for the study of the cellular dynamics of a range of signalling processes.
Introduction
The advance of miniaturized microfluidic systems for chemical and/or biochemical applications based on so-called Lab-on-a-Chip technology has demonstrated that such micro systems represent the ability to “shrink” conventional bench systems to the size of a few square centimetres with major advantages of speed, performance, integration, portability, reduced sample/solvent quantity, automation, hazard control and lower cost [1–6]. These merits are important for a variety of applications in analytical chemistry, biochemistry, clinical diagnosis, medical chemistry and industrial chemistry [1, 2]. Consequently, numerous micro total analysis systems (μ-TAS) and micro reactor systems have been developed, and many more are currently under investigation [2].
For the study of cellular and subcellular systems, a wide range of analytical methods have been used with fluorescence techniques being the most common. In addition, fluorescence-based imaging is a highly attractive methodology for the study of organelle dynamics, identifying subcellular compartments and monitoring biological kinetics [7]. Over the last two decades, Raman spectroscopy has become an increasingly important technology with ability to study the biophysics and biochemical processes involving cells [4, 8–12]. Since Raman spectroscopy is based on vibrational transitions where frequency shifts are associated with specific molecular vibrations within the sample of interest, it enables the identification of polarizable bio/chemical species, the elucidation of molecular structure and the investigation of interface reactions, all in a non-destructive manner. In addition, unlike fluorescence-based techniques, Raman spectroscopy does not require labelling dyes and since water is almost Raman “transparent”, the technique is ideally suited for analysing cell-based biological systems. Coupling a Raman spectrometer with a confocal microscope enables the acquisition of full spectral information with a high spatial (<1 μm) resolution in three dimensions. Raman spectroscopy, however, suffers from an inherent poor level of sensitivity compared for example to fluorescence, which can be 12–14 orders of magnitude more sensitive. As a consequence, despite advances in detector technologies, the technique can be less than ideal for the direct detection of intracellular components present at low concentrations, which may take from a few tens of seconds to a few minutes for spectral acquisition [12]. With the unexpected discovery of surface-enhanced Raman scattering (SERS) by Fleischman et al. [13], Raman intensity can now be dramatically increased (a factor of up to 105–1010) with the inclusion of metallic nano structures either on a substrate surface or in a colloidal solution [8]. For cellular and subcellular analysis with SERS, colloid nanoparticles (e.g. silver or gold) are normally loaded into cells by different means such as general incubation (fluid-phase uptake) or ultrasonication-assisted uptake [14, 15]. Due to its chemical inactivity, gold nanoparticles are generally regarded to be more suitable for incorporation within living cells [14].
To analyse living cells using Raman spectroscopy, the cells are usually fixed at a specific location. The most commonly used method for cell fixation is to seed or grow cells on a substrate, e.g. a microscope cover slide. Other approaches such as optical tweezers have also been reported [9]. However, the conventional batch operation in a static system limits the in situ Raman analysis, especially when a series of reagent treatments are required. With those limitations in mind, the development of microfluidic and associated Lab-on-a-Chip technologies provides unique opportunities for delivering and immobilizing cells on a microchannel surface, prior to introducing different reagents with a continuous flow in a given sequence [3, 4] whilst under spectroscopic investigation.
In this study, we report the successful integration of a range of techniques including microfluidics, surface-enhanced Raman scattering (SERS) and confocal microspectroscopy which enables in situ characterization of single living CHO cells with high spatial and temporal resolution. By using the microfluidic methodology, cells and reagents were introduced into the chip in a continuous flow as a series of plugs in a given sequence where agonist (ionomycin) evoked intracellular Ca2+ fluxes and the cell’s real time spectral response was recorded.
Materials and methods
Confocal Raman microspectroscopy
All Raman spectra were acquired with a LabRam inverted microscope spectrometer, manufactured by Jobin Yvon Ltd. Figure 1 shows the schematic of the experimental setup. The spectrometer was equipped with dual laser sources at wavelengths of 780 nm (diode laser, 70 mW) and 633 nm (He–Ne laser, 20 mW), confocal optics, a holographic transmission grating, and a charge coupled device (CCD) detector with 1,024 × 256 pixels. The instrument included a precision motorized X–Y sample stage for automated mapping at spatial resolution down to less than 1 μm and extensive software support (LabSpec 4.18) for data processing. In this study, an objective lens of ×50 magnification, 17-mm working distance and numerical aperture (NA) of 0.45 was used (L Plan SLWD 50, Nikon, Japan). This objective lens was mounted on a PI-721.10 piezo actuator (Physik Instrumente, Germany) for automatic focussing of the microscope objective at different depths in the Z direction enabling 3D mapping. A grating with 1,800 grooves mm−1, a confocal aperture of 300 μm and an entrance slit of 150 μm were selected for the experiments. The Raman spectrometer wavelength range was calibrated using the centre frequency of the silicon band from a silicon sample (520.2 cm−1). Using these conditions, a typical acquisition time of 1 s was used to collect SERS spectra from cells within the microchannel.
Fig. 1Schematic of experimental setup
Microfluidic device fabrication
The microfluidic device was constructed using a manifold clamping method according to published procedures with some adaptations [16–18]. The assembly of the device is illustrated in Fig. 2a. Briefly, the microchip consisted of a PARAFILM® sheet (thickness 130 μm, American National Can Company, US) with a channel network and two glass plates which sandwiched the polymer film. The Y-shaped channel network (Fig. 2b) cut through the film was 500-μm wide. The top glass plate (B-270, 25 × 25 × 3 mm) had three holes (diameter 1.5 mm) drilled through at appropriated positions in order to link the ends of the channels with inlet/outlet tubing. The bottom glass plate was a thin quartz coverslip (22 × 22 mm, Agar Scientific Ltd, UK) which had a thickness of 250 μm in order to minimise the glass background during Raman measurements. This sandwich chip was then clamped using two aluminium frames with screws. The windows on the frames were designed for tubing connections (through top frame) and for optical passage (through bottom frame).
Fig. 2(a) Assembly of microfluidic device and (b) Y-shaped channel network (channel depth 100 μm, width 500 μm) with cells loaded (c) for examination
Two KDS 200 syringe pumps (KD Scientific Inc., USA) were used to deliver cells in suspension and test solutions into the microchip channel (Fig. 2c). Ethylene tetrafluoroethylene (ETFE) polymer tubing with an inner diameter of 250 μm, on–off valves, and appropriate fittings and connectors, all obtained from Upchurch (Upchurch Scientific Inc., USA), were used for plumbing to link the chip and the syringes.
Cell culture and assay reagents
CHO-K1 (Chinese hamster ovary, Cricetulus griseus) cells were supplied by ATCC/LGC Promochem (ATCC® No. CCL-61™, LGC Promochem, UK). The cells were cultured routinely in DMEM/F-12 medium without L-glutamine (Invitrogen Ltd, UK) which was supplemented with fetal bovine serum (Invitrogen Ltd) to a final concentration of 10%, and L-glutamine (Invitrogen Ltd) to a final concentration of 4 mM. An incubator was used at 37 °C supplying 5% CO2. The concentration of cells used in loading the chips during this experiment was in the range of 7.5 × 106 cells mL−1.
A wash solution consisting of a modified Tyrodes buffer was used to wash the cells and to prepare the test solution. The Tyrodes buffer was composed of 145 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM D-glucose and 1.2 mM MgCl2. CaCl2 (99.5%, BDH AnalaR, 150 mM dissolved in Tyrodes buffer) and probenecid (98%, Sigma, 0.834 M dissolved in 1 M NaOH aqueous solution) were then added to the Tyrodes buffer giving final concentrations of 1.5 mM and 2.5 mM for CaCl2 and probenecid, respectively. Ionomycin test solutions were made by adding ionomycin stock (1 mM in DMSO) into wash solutions for a concentration of 50 μM. Ionomycin was obtained from Calbiochem (Calbiochem of EMD Biosciences, Inc., USA), and DMSO from Sigma–Aldrich (99%, D2650). The flow rate for the introduction of ionomycin test solution into the microchip was 2 μL min−1.
Gold colloid with a particle size of 50 nm (EM.GC50), suspended in water, was supplied by BBinternational Ltd, UK. The gold nanoparticles were introduced into cells by a passive uptake method where the cells were incubated with gold colloid solution at desired concentration (20% colloid solution, v/v) and room temperature for 50 mins. Prior to loading into the microfluidic chip, the cells were washed with wash solution to remove the culture media and gold nanoparticles outside cells. The cells were re-suspended in the wash solution for measurements.
Results and discussion
SERS effects
After loading cells into the microfluidic channel, Raman spectra were taken from a selected single cell (Fig. 2c). By using the near-infrared laser excitation (780 nm) the optical and thermal effects of laser illumination on the living cells were minimised [19, 20]. In addition, using a near-infrared laser can significantly reduce the fluorescence interference background and light scattering from the quartz base plate. It has been reported that silver or gold nanoparticles with an individual size in the range of 20–60 nm can yield significant enhancement of Raman scattering for cellular analysis when nanoparticles are introduced into cells [14]. This has been confirmed in this study by using gold nanoparticles with a size of 50 nm.
Figure 3 shows a typical spectrum (upper) taken from a cell incubated with gold colloid solution. The spectrum was recorded in the range from 300 to 2,800 cm−1 with an acquisition time of 1 s. We observed that with the introduction of gold nanoparticles the Raman spectra were clearly detectable under those conditions. For comparison, a spectrum obtained from a cell incubated in medium without gold colloid solution is also shown in Fig. 3 (lower). It can be seen that even though the spectra acquisition time was increased to 10 s it was still unable to obtain a good quality spectrum without gold nanoparticles present. This observation confirmed the significant enhancement of Raman scattering with the presence of gold nanoparticles which also enabled a fast spectra acquisition although the exact mechanism of the SERS enhancement of Raman signals is not fully understood [21]. The introduction of gold nanoparticles therefore significantly shortened signal acquisition times mainly owing to the increase in sensitivity which in turn allowed an array of spectra to be obtained in a relatively short time period.
Fig. 3Raman spectra from cells following incubation in medium with gold colloid (upper spectrum) and without gold colloid (lower spectrum)
Characterization of single living cells by 3D mapping
Using the SERS technique described above it was possible to obtain spectra from a single living cell which represented a “fingerprint” from which various chemical constituents in the cell can be assigned. Combined with an automated microscope stage which offers spatial resolution (i.e. 1 μm), mapping of an entire cell area at a specific plane was carried out. It has been suggested from previous studies that most of the spectral bands associated with living cells occur in the range 800–1,700 cm−1 [11, 22, 23], hence this range was used in this study for mapping. Figure 4a shows an “image” of Raman spectra obtained from the middle layer across the single cell by mapping an area of 21 × 21 μm2 with a spatial step of 1 μm in both X and Y directions. The mapping provided information on the distribution of selected bands, as seen in Fig. 4b in the range from 1,290 to 1,370 cm−1, which represent most of the significant bands associated with DNA and proteins within a cell’s nucleus and cytoplasm [11, 23].
Fig. 4a Spectral mapping of a single CHO cell on an X–Y plane and b corresponding spectra from three positions in the area of nucleus, cytoplasm and membrane, respectively
In general, the Raman spectra of single CHO cells showed contributions from all its cellular components including nucleic acids, proteins, lipids and carbohydrates. Table 1 summarises the band assignment for the Raman spectra taken from CHO cells based on the published data [8, 9, 12, 23–27]. Comparison of the spectra taken from different positions across the cell on an X–Y plane (Fig. 4) indicated that strong peaks from the nucleus spectrum corresponding to DNA sugar–phosphate backbone (895 and 1,142 cm−1), and bases G (1,320 and 1,487 cm−1), A (1,420 and 1,578 cm−1), T (1,176 and 1,376 cm−1) and C (1,420 cm−1) were noticeably reduced in the cytoplasm and membrane spectra (Table 1). This change was expected as the nucleus contains high densities of DNA, whilst the cytoplasm also had significant quantities of RNA contributing to the corresponding peaks. As expected, the spectrum taken from membrane area showed significant peaks corresponding to lipids (1,068 and 1,453 cm−1).
Table 1Band assignment for Raman spectra of CHO cellsBands (cm−1)AssignmentsDNA/RNAProteinsLipids830Phosphodiester BkB [12, 23, 24]Tyr [12, 23, 24]895Phosphodiester BkB, deoxyribose [12, 23, 24]940ν (C–C), α-helix [9, 12, 23]1,004Phenylalanine [9, 12, 23]1,065ν (C–O) [12, 23]ν (C–C) chian [25]1,126ν (C–N) BkB [9, 12, 23]ν (C–C) chain [25]1,144Ribose–phosphate [12, 23]1,157Ribose–phosphate [20, 26]1,176T, C, G [9, 12, 23, 24]Phenylalanine [12, 23]1,230C [12, 23, 24]1,266Amide III [25]δ (C=CH2) [25]1,295δ (CH2) [25]1,320G [24]1,342A [12, 23, 24]1,376T, A, G [9, 12, 23, 24]1,448δ (CH) [9, 12, 23]δ (CH) [9, 12, 23]1,482A, G [12, 23, 24]1,566Hemoglobin [25]1,578A, G, purine [12, 23, 24]1,603A, C [24]1,643Amide I [27]Abbreviations: BkB DNA sugar–phosphate backbone, Tyr tyrosine, A adenine, T thymine, G guanine, C cytosine, ν stretching vibrations, δ deformation vibrations
Using the confocal optics of the microscope system, the chemical concentration distribution in the Z axis at three levels in the nucleus was examined (Fig. 5) and indicated that the main peaks positions were generally identical but the peak heights were markedly different, indicating a concentration difference. From Fig. 5a significant a peak was observed at 1,320 cm−1 on the spectrum taken from Z = 3 μm (near to the bottom of the cell) which was assigned to DNA bases G [23], whilst the peak at 1,450 cm−1 from Z = 6 μm indicated a strong deformation from a C–H stretch in proteins [8, 12, 23]. Thus, by collecting spectra from different spatial positions, it provided an approach to build a 3D mapping of the distribution of chemicals within a single cell.
Fig. 5Raman spectra taken at three levels along the Z direction in the nucleus area
It should be noted that the spectra taken from different positions are distinct in terms of both Raman band amplitudes and band positions. This spectral variation can be attributed to the native chemical inhomogeneity within a cell. However, the possibility of non-uniform distribution of gold nanoparticles within the cell cannot be excluded [15]. In addition, this non-uniform distribution can also take place during the time-resolved monitoring because dynamic processes inside the cell can lead to local fluctuations of the particle densities and hence changes of the spectra. To further develop this spectroscopic technique for both qualitative and quantitative analysis, technologies need to be explored in order to deliver and position nanoparticles within cells in a controllable format for a uniform distribution. Nevertheless, it is still possible using this promising methodology to examine a specific point within a cell in a dynamic way with time, especially when applying stimulation, e.g. an agonist, under microfluidic control conditions.
In situ monitoring of cellular chemical dynamics
Finally, we monitored the chemical dynamics of the cell when exposed to the agonist ionomycin [3, 28]. After loading cells into the chip channel, a 40-min period of settlement was allocated before introducing the ionomycin solution at a constant flow rate of 2 μL min−1. Whilst focussed on a region close to the cell nucleus, a series of spectra were taken at an interval of 1 s in the range from 800 to 1,700 cm−1 (Fig. 6a). It can be seen from the spectra (Fig. 6b) that most of the peaks corresponding to nucleic acids, proteins and lipids remain visible with time but the peak heights vary noticeably, indicating the concentration change of these compounds. One of the most significant changes is the appearance of a peak at 1,643 cm−1 which can be assigned to amide I [27]. The time profile of this peak (Fig. 6c) reveals the concentration change of amide I within the cell, showing a similar trend to that of the Ca2+ flux evoked by the agonist ionomycin [3] which is commonly used in biomedical research to stimulate the intracellular production of proteins such as interferon [29]. Since amide I contributes spectrally to the Raman spectrum of interferon, it follows that the in situ monitoring of such molecules could in future be used to produce characterization of protein expression dynamics at subcellular levels. Clearly, more studies are required in order to understand the mechanism and cause of the concentration change of amide I within the cell when exposed to agonist ionomycin.
Fig. 6In situ monitoring of cellular dynamics by recording a series of Raman spectra (a) from a specific area at an interval of 1 s. The first spectrum, at time 0, was taken when no agonist was introduced (b); (c) shows the time profile of the peak at 1,643 cm−1 corresponding to the change of amide I
Conclusions
The SERS technique has been used for characterisation of single CHO (Chinese hamster ovary) cells with a microfluidic device where gold nanoparticles were introduced into cells for Raman enhancement. The use of an inverted microscope optical systems in combination with a charge coupled device (CCD) detector allows the measurement of Raman spectra for simultaneous analysis of bio/chemical species within cells. In addition to a precision automated X–Y sample stage, the confocal optics provides discrimination between points of different depths within the cell, enabling chemical mapping in three dimensions. By using microfluidic methodology, the cell manipulation and reagent delivery were performed in a controllable manner. By introducing cells and test reagent into the chip in a continuous flow as a series of plugs in a given sequence, it enabled the in situ chemical characterization of single CHO cells with a high degree of spatial and temporal resolution. This allows the real time monitoring of the dynamics of the agonist-evoked Ca2+ flux response. The approach described has the potential to be used for the study of the spatial dynamics of a range of intercellular processes.

surface-enhanced raman scattering (sers)


Setting the Public Agenda for Online Health Search: A White Paper and Action Agenda

Background Searches for health information are among the most common reasons that consumers use the Internet. Both consumers and quality experts have raised concerns about the quality of information on the Web and the ability of consumers to find accurate information that meets their needs.
Introduction
Searches for health information are among the most common reasons that consumers use the Internet. The Pew Internet & American Life Project (Pew) reported in 2003 that 80% of Americans with Internet access have used the Web to get health or medical information [1]. The Internet has transformed the ability of consumers to find health information and to connect with other individuals with similar interests. The Internet has been recognized as an important source of health information by the federal government, which established a series of goals relating to access and quality of information on the Internet in the Healthy People 2010 action plan [2].
Health information on the Internet can dramatically improve consumers' health-care and lifestyle choices. However, increased access to Web-based information has also raised concerns about the quality of information consumers are using, and the impact of this information [3]. Disparities in access to information have also become apparent. These factors suggest the need to better understand how consumers find health information on the Web, how to evaluate the quality of information retrieved, and how to help consumers to critically evaluate and manage information. These factors suggest the need to better understand how consumers find health information on the Web, how they evaluate the quality of information retrieved, and how to they could be helped to critically evaluate and manage information.
Research on health Web sites raised concerns about the quality of information on the Web [4]. A 2001 study by RAND for the California Healthcare Foundation showed that information on health Web sites is often incomplete or out of date [5]. This might be of little concern if consumers routinely consulted health-care professionals about the information. However, Pew found that 69% of consumers did not discuss the information they found with a doctor or nurse.
Many people use search engines to find the information they use to help make personal health decisions. Search engines and the Internet have vastly improved access to health information for many consumers. However, search processes and results vary considerably among search engines, and are not transparent to consumers. The criteria used to identify and rank health-related Web sites vary among search engines, and often is not apparent to consumers. Search results may be affected by the structure of content on health Web sites, consumer search terminology, and the use of paid placements by the search engine.
In short, research on health searches suggests that the process by which consumers locate health information on the Internet, and the evaluations they make regarding which Web sites to review are important variables in the quality of information they ultimately view and use. Improved understanding of factors influencing online searches will facilitate technical and educational approaches for maximizing quality and benefit of health searches.
Methods
In 2003, URAC and Consumer WebWatch (CWW), a project of Consumers Union, carried out a project funded by the Robert Wood Johnson Foundation to examine factors influencing the results of online health searches and to develop an agenda for future research and development that would improve the results of health searches. We reviewed published literature and industry reports, and convened two stakeholder groups consisting of consumers, quality experts, search engine experts, researchers, health-care providers, informatics specialists and others.
Literature Review Method
Our literature review was not exhaustive: its purpose was to provide a baseline understanding of consumer, Web site, quality measurement, and search engine factors that influence the results of searches for health information. We conducted a search of key terms in the Cumulative Index of Nursing and Allied Health Literature (CINAHL), Medline, PubMed, Expanded Academic ASAP, Lexis-Nexis, Proquest, Ingenta, and related databases in health care, information science, and computer science. The initial searches took place in early 2003, but citations were added as they were identified.
Where initial searches revealed poor topic coverage, associated reference lists, books and other media that were considered to inform the topic were included. The following search terms were included: Web-based, Web site, information quality, Web search, consumer health, eHealth, health information, search engine, information retrieval, information seeking. We also examined bibliographies of articles retrieved by electronic searches and solicited recommendations from members of the project advisory committee. We discontinued searching in specific topic areas when project staff believed we had adequately described current understanding of key issue areas.
Methods for Convening Stakeholder Meetings
An open announcement about the project and recommendations from industry leaders helped identify interested stakeholders, and participants were selected by URAC and CWW with guidance from a project advisory committee. Not everyone invited was available to attend. We attempted to achieve a balance of different stakeholders at each meeting. Meetings were held in California and Washington, DC to facilitate participation.
The purpose of each stakeholder meeting was to review existing knowledge about results of consumer searches for health information, and to develop recommendations for additional research, technical improvements, and educational approaches needed to improve the results of online consumer searches for health information. Participants reviewed the summary recommendations presented in this article after the meeting and had the opportunity to comment, but were not asked to vote on or endorse the recommendations.
For the purposes of this project, we assumed that most searchers would prefer information that is accurate and reliable. These attributes are also components of effective health communication [2]. This was our working definition for quality Web sites. The perception of other elements that might be used to define quality, such as the site's reading level and comprehensiveness, will vary depending on the user and the user's needs at a given time.
Results
Results of Literature Review
How Consumers Use the Internet to Locate Health Information
An April 2003 report from the Pew Internet & American Life (Pew) report provided an overview of the US Internet consumer population [6]. The study found that Internet access has grown across-the-board, but that demographic gaps remain. A variety of factors continue to separate Internet users from non-users. Internet users tend to be younger and more affluent, and are more likely to be employed, white, well-educated, and to be suburban or urban residents.
Pew noted that consumers often overestimate their knowledge of the Internet and their ability to locate information. A 2002 analysis by Houston et al using Pew data noted a need to educate patients about searching for health information online and for tools to help them identify high quality information [7]. They also found that chronically ill Internet users were often relatively new to the Internet, but noted that they were more likely than those in good health to discuss findings with their physicians.
Consumer Search Strategies
A 2002 Pew Internet & American Life Project poll found that the typical health-information seeker usually starts searching for medical information at a general search site, not a medical site. Eighty-one percent of online health seekers start at a search engine or use the search function of a general portal such as the Yahoo home page, MSN, or AOL. Consumers visit two to five sites during an average visit and typically spend at least thirty minutes on a search [8].
Several studies have investigated behaviors consumers exhibit in retrieving and health information on the Internet and in assessing its quality. Eysenbach and Köhler, examining Web searchers in Germany, found that although search technique was often suboptimal, Internet users found the health information they were looking for relatively quickly [9]. A search optimization firm, iProspect, reports that users generally use the same search engine for all types of search requests. Users look at up to three pages of search results to determine relevance, and abandon a search if they do not find appropriate results in the first three pages. Users usually modify their query after abandoning an initial search, and may at that point change search engines [10]. These findings illustrate the importance of search engines to the process of retrieving health information. They imply a business rationale for search engines to ensure that health searchers locate what they want, since they may otherwise lose search traffic.
Comprehension, Literacy, and Access Issues
Searches are heavily influenced by the search terms used, even when the terms used are considered to be synonyms. Use of lay terminology for a health subject can result in unrelated or misleading information [11]. Berland et al concluded that accessing health information using search engines and simple search terms was not efficient because Web sites are inconsistent in their provision of key information, and because high reading levels are required to comprehend Web-based health information [4]. Also, the relevance of information located was often of limited value, which may have been due to terminology used in the original search phrase. Non-English speakers face challenges finding and reviewing information on the Internet. One Internet accessibility study for people with disabilities found that there are significant access barriers. Governmental and educational health-information Web sites were more accessible than other categories, such as Web portals and community sites [12].
Physician Responses to Internet Information
A study of physician views on online information found that physicians increasingly encounter patients who have conducted health searches. Use of the Internet by patients does appear to affect treatment processes: for example, many physicians reported having changed the treatment protocols they had initially planned as a result of consumer requests. Although most physicians believe the information their patients find is accurate, many believe that having to discuss this information with their patients decreases their efficiency and challenges their authority. Some are also concerned that the information may be inaccurate. The study concluded that quality of information on the Internet is critical, as it does influence both patient requests and physician treatment choices [13]. In an effort to steer patients to credible Web sites, some health-care organizations have begun to suggest ( "prescribe") credible Web sites to their patients in the course of their consultations [14].
Consumer Evaluation of Web Site Credibility
Experts and consumers use different criteria for evaluating the quality of Web sites. Eysenbach found that consumers assessing the credibility of a Web site primarily looked for the source, a professional design, a scientific or official touch, language, and ease of use. Study participants never checked any "about us" sections of Web sites, disclaimers, or disclosure statements. Very few participants noticed and remembered which Web sites visited [9]. A Consumer WebWatch (CWW) study of consumers reported findings similar to Eysenbach's: once people get to a site, they do not use rigorous criteria to assess the site's credibility. For example, they almost never referred to a site's privacy policy. The average consumer paid far more attention to the superficial aspects of a site, such as visual cues, than to its content. Nearly half of all consumers in the CWW study assessed the credibility of sites based in part on the appeal of the overall visual design, including layout, typography, font size, and color schemes. In comparison, a parallel group of health and finance experts were far less concerned about the surface aspects of these industry-specific types of sites and more concerned about the breadth, depth, and quality of a site's information [15].
How Web Sites Influence Availability of Quality Health Information
Techniques for Conveying Information about Web Site Content
The structure of a Web site influences how information can be retrieved from the site by a search engine, as well as the usability of the site for consumers. Coding and structure of Web sites can facilitate retrieval by search engines or can pose a barrier to information retrieval. Coded information on a Web site is processed through the search engine algorithm, and determines whether and how the site is ranked in search returns. The same tags and codes that can be used to highlight information on a legitimate Web site may also be used by "spoofers" who try to lure traffic onto the site.
In general, Web sites can support retrieval of information on their pages by using metadata, metatags and keywords to guide search crawlers to important content. These codes provide a means for relaying information directly to the search engine. Keywords are recognized indicators of specific services or products that can be used to increase specificity of searches and help Web sites attract "qualified" traffic. One strategy for enhancing search rankings of quality Web sites is to code certain types of information for consistent retrieval by the search engine. Efforts are under way to implement metadata codes to support a "semantic Web." The semantic Web uses code to establish relationships between words to enable search engines to effectively understand intent, rather than simply identifying the presence of a search term [16].
Quality Indicators for Web Site Content
Eysenbach et al found wide-ranging differences in studies of the quality of health Web sites. There are significant variations in study methods and rigor, quality criteria, study population, and topic chosen. Operational definitions of quality are often inconsistent. As a result, the conclusions on quality of health-related Web sites vary widely. Eysenbach found that the most frequently used quality criteria include accuracy, completeness, readability, design, disclosures, and references provided [16].
Griffiths and Christensen evaluated the quality of Web-based information on treatment of depression to identify potential indicators of content quality, and to establish whether accountability criteria are indicators of quality [17]. They found that although the sites examined contained useful information, their overall quality was poor. Sites typically did not cite scientific evidence in support of their conclusions.
Researchers have also studied the correlation between popularity of Web sites and quality of content. Meric et al found that more popular breast cancer-related Web sites were more likely than less popular ones to contain information on ongoing clinical trials, results of trials, and opportunities for psychosocial adjustment. These characteristics were also associated with a higher number of links. More popular sites were more likely to provide updates on other breast cancer research, information on legislation and advocacy, and a message board service. Measures of quality such as display of authorship, attribution or references, currency of information, and disclosure did not differ between popular and less popular sites [18]. In similar findings, Kunst et al found that while there is a correlation between credibility features and accuracy of information, the association is relatively weak [19].
These findings suggest that additional research is needed to identify indicators of content quality, and to correlate consumer preferences to quality indicators. Sites that include content correlated with popularity may best meet the public's desire for health information. Current search algorithms may not be in agreement with quality clinical indicators and performance measures currently used throughout the health-care industry.
Codes of Conduct
A wide range of tools has been developed to assist site developers to produce good quality sites and for consumers to assess the quality of sites. Adherence to accepted codes could theoretically be used as a factor in searches. Ratings instruments include codes of conduct, quality labels, user guides, filters, and third party certification. The value of these tools is unclear: studies have demonstrated that consumers do not routinely seek out information on certifications or adherence to voluntary codes. However, it is assumed by many that such codes benefit consumers indirectly by influencing Web site behaviors and practices. For example, most standards require sites to implement privacy protections and disclosure of site information as consumer protections. No research has been done on the effect of compliance to a code of conduct on Web sites.
A number of organizations have developed quality criteria for health-related Web sites, some with verification and some completely voluntary. Voluntary, self-certifying standards have been developed by the eHealth Code of Ethics of the Internet Health Coalition [20], the American Medical Association [21] and the Health On the Net (HON) Foundation [22]. URAC has developed a health Web site accreditation program that involves independent verification of compliance with its standards. URAC accreditation includes review of the Web site by an external auditor [23].
Web Site Rating Instruments
Web site ratings could be potentially be used to inform searchers and search engines as well, if ratings could be clearly correlated to quality. Two common approaches to rating Web sites include expert ratings, and user (consumer) ratings. Gagliardi and Jadad conducted two evaluations of Web site rating tools, published in 1998 and 2002 respectively [24,25]. They concluded that ratings instruments tend to proliferate and disappear, and that few have been validated for direct correlation between standards and quality. Few provide details on how they were developed, or provide instructions for use, or information about the inter-observer reliability and construct validity of the measurements.
Kim et al reviewed published criteria for evaluating health-related information on the Web, and identified areas of criteria-based consensus [26]. They identified 29 published rating tools and journal articles that had explicit criteria for assessing health-related Web sites. The most frequently cited criteria were those dealing with content, design and aesthetics of site, disclosure of authors, sponsors or developers, currency of information (includes frequency of update, freshness, maintenance of site), authority of source, ease of use, and accessibility and availability.
A number of tools are available to guide users in evaluating information on the Web. Interactive user guidance systems can be used to assess characteristics of Web sites. Tools such as DISCERN and QUICK allow Web site users to assess Web site credibility by responding to a series of questions [27]. Other organizations such as the National Library of Medicine, which operates MEDLINEplus, and the Medical Library Association, have developed guidelines and tips for consumers to evaluate health Web site content [28]. The European Union sponsored a collaborative project called MedCERTAIN to develop a rating system to enable consumers and professionals to rate quality information on the Web. The MedCERTAIN project evolved in a project called MedCIRCLE, which has developed a metadata coding language to mark quality indicators on health Web sites [29].
Discussion
Search Engines and Mediators of Health Information
Electronic and Human Mediation
Search engines serve an essential function in enabling users to find relevant information on the Internet. Recognizing the challenges of sorting the enormous amount of information on the Internet, many organizations are augmenting or mediating the results of electronic searches. Mediation can be either electronic or human-augmented techniques for reviewing information and making a pre-selected set of information available to consumers. One challenge to search engines and human mediators is making access to personalized information as effortless as possible, as consumers rarely use even the advanced search features currently available to them [30].
How Search Engines Work
Search engines and Web directories play a central role in facilitating access to health information. Web directories are organized Web site listings put together by human reviewers. Search engine listings are put together by automated systems and lack a navigable structure. Directories usually concentrate on indexing Web sites, while search engines typically index individual Web pages. Consumer searches for keywords will result in a valid match only if the keyword appears in the Web site's description. Hybrid models of search engines and directories are common.
Search Engine Indexing and Retrieval Methods
Virtually all commercial search engines rely on large powerful databases that utilize automated search agents called robots ("bots"), crawlers, or spiders. Search agents crawl the Web continuously to index information on Web sites. Crawlers capture metadata, page titles and textual content, and add them to the search engine's index or main database. The search engine's algorithm compares indexed data to the user term to process a search. Search engine algorithms are quite complex and scientific. They make frequent use of complementary directories aimed at optimizing and positioning Web sites in the right categories. Search algorithms are closely guarded as proprietary corporate information [31].
Current metrics for evaluating search engines include initial page retrieval capacity and the ability to revisit Web sites to update information. Currency of information, as demonstrated by elimination of non-working links to Web sites is also a performance metric. These criteria are features of business performance, not necessarily the content relevance or quality of the sites returned by a search.
Content and format of Web sites determine how they are indexed by search engines. Some search engines use keyword location, frequency, phrasing, and density as indexing and ranking factors. Type and number of links associated with a Web site are common indexing factors. Web sites also use tags to identify certain types of information. Search engine databases include only Web sites that have been registered with or indexed by the search engine-hence the importance of Web site developers making their sites accessible to automated agents, or becoming known to directory developers.
Ranking and Ratings
Ranking of sites in the final display of search results is of great importance to Web sites, users, and search engines. Ranking effectively drives the likelihood of particular sites being recognized and visited because, as noted, consumers rarely look at more than three pages of results. A poorly designed or executed search may produce an unwieldy list of Web site results that is difficult to navigate. Alternatively, searches that are too narrowly drawn may omit important sites.
Paid preference and placement by search engines also affects which sites are retrieved in a given search [32]. A study by CWW demonstrated that consumers experience considerable confusion about paid listings, and may not distinguish them from other returned listings [33]. The Federal Trade Commission has also expressed concern about how paid placement is disclosed to consumers, and has warned search engines to clearly distinguish advertising from search returns. Search engines may operate their own paid placement programs or obtain search results from third parties, who in turn operate paid placement programs.
Mediated Searches
Mediated searches may be as simple as having a librarian assist with a search, or they may be based on much more complex algorithms. Participants in the URAC/CWW stakeholder group noted that medical and general librarians play an important role in helping large segments of the population retrieve online information and learn effective search strategies. More complex mediated search strategies employ both human mediation and electronic queries to interface with users and focus a search.
Many search engines offer filters that allow users to exclude unwanted search results, most typically pornographic sites. Users, including libraries, can also install blocking software to prevent unauthorized use. However, this electronic mediation may unintentionally block desired health information and create an access barrier. For example, because pornography-blocking software and filters cannot perfectly distinguish between pornographic and non-pornographic Web sites, such products may block access to legitimate health-information sites, particularly those related to sexuality [34].
Gateways employ filters, either electronic or human, to accept or reject types of sites of information based on preset criteria. Gateways are used to organize information on the Internet through selection of resources based on quality and relevance of information to a particular audience. Internet resources are reviewed, classified, and stored with descriptive information. In the US, healthfinder.gov®, is a widely used gateway to selected consumer health and human services information resources provided by US government agencies and other organizations serving the public interest [35].
Participants in the stakeholder meetings noted that domain name extensions such as .com or .org could be used as a distinguishing feature of Web sites for the purpose of focusing search efforts. The World Health Organization is considering the feasibility of requesting a "dot health (.health)" extension for a pre-selected set of trusted Web sites [36]. In informal proposals describing the .health domain name, the extension is proposed for health information, services and organizations under a framework promoting minimum standards of conduct. Oversight of Web sites would be delegated to independent verifying organizations. The advantage to sites for adhering to standards of content quality would be more ready identification of sites by search engines as a result of the .health domain name.
Stakeholder Discussion of Literature Review
Research Needs to Address Consumer Evaluation of Web Quality
There is great variation in how consumers seek information via the Internet, and in how successful they are in searching for health information. Since there is significant consumer-level variation in how consumers search for health information, search algorithms that support variation and still return expected results will meet consumer needs most effectively. Additional research is needed on information needs of different consumer segments and how to effectively educate differing consumer segments to improve the results of their health searches. Research is needed on how to efficiently validate the quality of Web sites and communicate this information to consumers.
Research Needs for Web Site Quality Indicators
There is a need for tools to enhance recognition of quality Web sites by consumers and search engines. Such tools may be implemented by Web sites themselves, for example through increasingly sophisticated coding to highlight quality indicators. The MedCERTAIN project has been created precisely to address this issue, and has developed the HIDDEL vocabulary to mark features of Web sites [29]. Technical tools can be used to direct consumers more effectively to relevant, high quality information. In addition, since there are currently multiple tools for either self-evaluation or third party evaluation of Web sites, future research should be undertaken to validate these tools.
As noted, gateways filter information to increase its relevance to consumers and provide expert assessment regarding validity of sources is available. It may also be useful to develop more sophisticated search models for providing useful and relevant information to consumers via customization approaches. Such approaches could potentially be embedded in search algorithms. In addition, more research is needed on the impact of Internet-based health information on outcomes. The benefits and risks of health information, both from a health outcome and a system outcome (quality, cost), are poorly understood and should be examined further.
Research Needs for Search Factors Influencing Search Results
Search engines are increasingly important as a tool for locating and organizing information from the vast Internet resource. The volume of information on the Web is so significant that consumers may need different types of mediators, such as search engines or librarians, to help manage the volume of information. Human assistance is also helpful to counteract electronic spoofing and to help consumers overcome limitations in their search strategies.
To effectively improve health searches, more information is needed about search algorithms and how quality factors are identified in the algorithms. Search engines are also developing technology to search for synonyms, which may enhance health searches conducted by laypersons. It may also be helpful for search engines to develop methods to distinguish health related searches from other types of searches, rather than using a simple word match. Search technology to intuit consumer needs more effectively and learn from repeated searches could help search engines steer consumers to quality results. New technologies may ultimately be more effective than electronic filtering, requiring consumers to apply filters, or modifying their search strategies.
With technology advances, search engines may be able to identify quality proxies that could improve page rankings of high-quality Web sites. Search engines could, for example, give higher ranking to "official sites" for diseases. They could also piggyback onto credibility assessments provided by groups such as healthfinder.gov, or give higher ranking to sites listed in directories from trusted independent sources. Ultimately, adoption of technological solutions depends on the ability of researchers to understand the relationship between electronic proxies for quality and actual quality of content.
Discussion of Stakeholder Recommendations for Next Steps
The URAC and CWW expert panels discussed consumer, Web site, and search engine factors that influence the outcomes of health searches. In the course of discussion, they developed a number of recommendations for future research and development (Textbox 1). Their recommendations fell into several categories: needs for health services research, consumer and provider education, technological improvements, and development of tools and information to improve the results of health searches. For some recommendations, the evidence base for implementation is strong; for others, not. Implementation of some recommendations will be enhanced by creation of a national research agenda for health information and targeted funding to study and improve consumers' ability to locate and retrieve quality health information on the Internet. Other recommendations could be embraced at any time by researchers, educators or technology organizations as a business need becomes increasingly evident.
Recommendations of the Group
Leadership for Health Search Improvement
Organizations concerned about the quality and accessibility of health information online should continue to collaborate to promote "health search literacy."Collaborators should convene a leadership summit on health search literacy to discuss feasibility and implementation of many of the recommendations herein.Collaborating organizations shouldwork with funding organizations to develop a comprehensive long-term research agenda to improve health searches and increase access to quality health information;develop enhanced research methodologies to evaluate the quality, impact, and effectiveness of online health information.
Consumer-directed Tools
Create tools to support consumer health-information needs, including preset, prescreened health bookmarks and more guidance on how to reach health gateways and portals containing trusted health content.Develop and circulate a public domain brochure on health search strategies that could be branded and distributed by physicians, employers, health plans, and others to educate consumers.Develop public domain interactive, validated search strategy content pages that could be branded and used by health Web sites.
Research Needs
Identify the search needs and capabilities of diverse populations of searchers, including culturally diverse users and searchers with health needs of differing intensity and severity.Develop more understanding about how consumers interpret online health information, assess its credibility, and make health-related decisions.Research the relationship between consumer search strategies and consumer expectations for results to determine effective approaches for conveying information on the Internet.Research factors affecting physician assessments of Web-based information and how quality content affects physician recommendations to patients about online health-information resources.Assess the relationship between expert accreditation, quality seals, ratings and content quality, as well as the impact of such endorsements on both consumer behavior and Web site behavior.Research the correlation between Web site traffic volume and consumer satisfaction, particularly for health Web sites where there is variation in dimensions of quality such as accuracy, comprehensiveness, ease of navigation, and reading level.Evaluate content quality of Web sites in different domains, (eg, .gov, .edu, .com, and .org) to identify similarities and differences related to quality within and across categories of Internet domain namesEvaluate the impact of Internet-based health information on health outcomes: utilization, behavior change, knowledge, burden of illness and disease, or other measures.Research the relative effect of each component of a search algorithm (word frequency and placement, links, etc) for finding health information.Validate elements of some search algorithms, such as link frequency, as indicators of value/quality.Conduct periodic studies to monitor changes in accuracy and quality of content over time, including updating findings from the California HealthCare Foundation /RAND study [5].
Education Agenda
Develop models for offering health search education at teachable moments and in diverse consumer settings.Promote dissemination of existing educational tools and resources to assist consumers in evaluating health information on the Web more effectively.Develop user-appropriate tools and approaches to assist Internet users with special needs. High priority user groups may include disability, low literacy, and non-English speaking groups.Urge provider organizations to educate provider members on the value of offering Internet information and interactive learning recommendations as part of the therapeutic intervention.Educate health Web site developers on how to make information easy to find and how to meet the content-level of their intended users.Urge education organizations, in collaboration with health organizations, to develop a school-based or publicly available health search curriculum.
Technology Improvement Agenda
Continue to develop interactive features on search engines and sites to customize and personalize health searches.Develop more functionality for search engines to enhance selected health queries by offering additional relevant information.Develop technological markers or indicators that could be uniformly applied by Web site developers to indicate accuracy and comprehensiveness of health Web sites.Develop codes to indicate when information on a Web site supercedes previous information.Develop collaborations between health quality and search engines experts to develop codes for validated quality proxies.Develop search technology similar to that used in the commercial sector to direct consumers to related, relevant information based on both searching and viewing behaviors.Enhance personalized searches by building search engine capability to "learn" from repeated searches and user behavior.
Expanding the Market for Quality
Develop a health equivalent of "BizRate" or "eBay" surveys that can be used by consumers to evaluate Web sites after viewing. Existing models for such a survey could be adapted and disseminated.Sponsor a competition for individuals or organizations to design a search algorithm that returns the most credible health results as evaluated by experts. Design a separate contest for the most effective business plan to make the business case for building quality factors into health searches.
Conclusion
The Internet has opened a vast library of information to consumers of health information and made that information more accessible than ever before. This represents a significant step forward for consumers. However, the volume of information and the variable quality of information has created new interpretive challenges. Now, one great challenge is helping consumers find the information they want that is also accurate, reliable, and presented in an accessible format. Searches for health information rely on a complex interplay of search algorithms, Web site content and coding, and consumer behaviors. The recommendations presented here address each of those factors with ideas for further research as well as more immediate recommendations for action. This agenda is a start at maximizing the potential of the Internet to deliver high-quality health information for diverse users.

internet
ehealth
health services research
information management
quality of health care
consumer participation
patient education